WO2023231149A1

WO2023231149A1 - Recombinant orthohepevirus genotype c1 orf2 protein and preparation method therefor and use thereof

Info

Publication number: WO2023231149A1
Application number: PCT/CN2022/105382
Authority: WO
Inventors: Wenshi Wang; Hongbo Guo; Renxian TANG; Kuiyang ZHENG; Hongjuan YOU; Xiaohui DING; Jikai Zhang
Original assignee: Xuzhou Medical College
Current assignee: Xuzhou Medical College
Priority date: 2022-05-25
Filing date: 2022-07-13
Publication date: 2023-12-07
Anticipated expiration: 2024-11-25
Also published as: CN114957411B; CN114957411A

Abstract

The present invention provides a recombinant Orthohepevirus genotype C1 ORF2 protein and a preparation method therefor and use thereof, and belongs to the technical field of hepatitis E virus. A recombinant rat HEV-C1 ORF2 protein provided by the present invention has an amino acid sequence set forth in SEQ ID No. 1, the recombinant rat HEV-C1 ORF2 protein can retain immunogenicity of rat HEV to the utmost extent, and the recombinant protein can stimulate different species of animals to produce specific antibodies. HEV-C1 ORF2-targeted specific antibodies generated in serum of rats immunized with HEV-C1 ORF2 protein can effectively block specific binding of HEV-C1 ORF2 virus-like particles to rat hepatoma cells RH-35, and thus the recombinant protein is proved to be capable of being used for preparing HEV-C1 vaccines.

Description

RECOMBINANT ORTHOHEPEVIRUS GENOTYPE C1 ORF2 PROTEIN AND PREPARATION METHOD THEREFOR AND USE THEREOF

TECHNICAL FIELD

The present invention belongs to the technical field of hepatitis E virus, and particularly relates to a recombinant Orthohepevirus genotype C1 ORF2 protein and a preparation method therefor and use thereof.

BACKGROUND

Hepatitis E Virus (HEV) is a non-enveloped, positive single-stranded RNA virus that has been classified into the Orthohepevirus of the family Hepeviridae (Smith DB, Simmonds P, Members Of The International Committee On The Taxonomy Of Viruses Study G, Jameel S, Emerson SU, Harrison TJ, et al. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2014 Oct; 95 (Pt 10) : 2223-32) . The genus Orthohepevirus includes four types of HEV-A to HEV-D (Smith DB, Simmonds P, Izopet J, Oliveira-Filho EF, Ulrich RG, Johne R, et al. Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol. 2016 Mar; 97 (3) : 537-42) , wherein the HEV-A primarily infects humans and causes hepatitis E infection in humans, while HEV-C1 primarily infects rats (Reuter G, Boros A, Pankovics P. Review of Hepatitis E Virus in Rats: Evident Risk of Species Orthohepevirus C to Human Zoonotic Infection and Disease. Viruses. 2020 Oct 9; 12 (10) ) , and is called rat HEV (rHEV) . In 2010, the rat HEV was first found in wild rats in Germany (Johne R, Heckel G, Plenge-Bonig A, Kindler E, Maresch C, Reetz J, et al. Novel hepatitis E virus genotype in Norway rats, Germany. Emerg Infect Dis. 2010 Sep; 16 (9) : 1452-5) , and then found by isolation in Europe, Asia and the United States (Johne R, Dremsek P, Reetz J, Heckel G, Hess M, Ulrich RG. Hepeviridae: an expanding family of vertebrate viruses. Infect Genet Evol. 2014 Oct; 27: 212-29) . Previous studies have shown that rHEV has a very low probability of developing cross-host infection. However, in 2018, the rat HEV viral genome was found in a liver transplant patient. Subsequently, the rat HEV was found to cause acute hepatitis in immunodeficient patients and cases of human infected with rHEV were found in Hong Kong (China) , Spain and UK (Sridhar S, Yip CC, Wu S, Chew NF, Leung KH, Chan JF, et al. Transmission of Rat Hepatitis E Virus Infection to Humans in Hong Kong: A Clinical and Epidemiological Analysis. Hepatology. 2021 Jan; 73 (1) : 10-22. ; Sridhar S, Yip CCY, Wu S, Cai J, Zhang AJ, Leung KH, et al. Rat Hepatitis E Virus as Cause of Persistent Hepatitis after Liver Transplant. Emerg Infect Dis. 2018 Dec; 24 (12) : 2241-50) , which shows that the rat HEV can infect humans and is zoonotic. Rat HEV, like other HEV viruses, contains three Open Reading Frames (ORFs) encoding ORF1, ORF2 and ORF3 proteins, respectively. ORF1 is a non-structural protein and is involved in viral replication. ORF2 gene encodes the viral capsid protein ORF2, which is primarily involved in assembly of viral particles and binding to a host cell and inducing production of neutralizing antibodies by the host. ORF3 gene encodes multifunctional phosphoprotein ORF3, and is primarily involved in the release of virions. Rat HEV is capable of encoding ORF4, and its C-terminus overlaps that of ORF1 and its function is unknown.

ORF2 has three different forms, namely infectious ORF2, glycosylated ORF2 and truncated ORF2, wherein the infectious ORF2 is mainly present in the viral particles, and the glycosylated ORF2 and truncated ORF2 are the major secreted forms of ORF2 (Yin X, Ying D, Lhomme S, Tang Z, Walker CM, Xia N, et al. Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection. Proc Natl Acad Sci U S A. 2018 May 1; 115 (18) : 4773-8) . ORF2 capsid protein is highly conserved and has high immunogenicity. ORF2 is capable of stimulating the body to generate humoral immune responses, and is also a primary target for cellular immune responses. The corresponding antibodies generated by ORF2 stimulation can be detected in the serum of the patient.

The HEV-A and HEV-C1 have a distant genetic relationship, and the homology of ORF2 of the two is only 50%–60%. This suggests that the difference between the immunogenicity of human ORF2 and that of rat ORF2 is large, and thus the cross reactivity between the ORF2 protein of the human HEV and an ORF2-targeted antibody generated after the rat HEV infects a human body is low, and the ORF2 protein of the human HEV can neither be used for detecting a serum sample of a patient contracting the rat HEV nor be used for developing a vaccine against the rat HEV. In conclusion, the research and development of the recombinant rat HEV ORF2 protein is of great importance to such prevention and treatment strategies as detection means of rat HEV and vaccine preparation.

SUMMARY

In view of this, the present invention aims to provide a recombinant Orthohepevirus genotype C1 ORF2 protein and a preparation method therefor and use thereof. The recombinant rat HEV-C1 ORF2 protein is a consensus amino acid sequence designed according to the known ORF2 of rat HEV, and can retain immunogenicity of the rat HEV to the utmost extent; the recombinant protein can stimulate different species of animals to produce specific antibodies.

The present invention provides a recombinant rat HEV-C1 ORF2 protein, wherein an amino acid sequence of the recombinant protein is set forth in SEQ ID No. 1;

or the amino acid sequence of the recombinant protein has at least 90%identity to a sequence set forth in SEQ ID No. 1.

The present invention provides a gene encoding the recombinant rat HEV-C1 ORF2 protein, wherein a nucleotide sequence of the gene is set forth in SEQ ID No. 2.

The present invention also provides an expression vector of a recombinant rat HEV-C1 ORF2 protein comprising a plasmid backbone and the gene.

Preferably, the gene is inserted into a multiple cloning site of the plasmid backbone. The present invention also provides a recombinant bacterial strain expressing the recombinant rat HEV-C1 ORF2 protein, wherein the recombinant bacterial strain is transformed with the expression vector.

Preferably, E. coli BL21 is used as host bacteria for the recombinant bacterial strain. The present invention provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a vaccine for preventing human infection of rat HEV.

Preferably, the vaccine is a subunit vaccine.

The present invention also provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a reagent for detecting human infection of rat HEV.

The present invention provides a monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein.

The present invention provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a reagent for detecting rat HEV.

The present invention provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a vaccine for preventing human infection of rat HEV.

Compared with the prior art, the present invention has the following beneficial effects: the recombinant Orthohepevirus genotype C1 ORF2 protein provided in the present invention is a consensus amino acid sequence designed according to the known ORF2 of rat HEV, and can retain immunogenicity of the rat HEV to the utmost extent; the recombinant protein can stimulate different species of animals to produce specific antibodies. Through ELISA assay, the recombinant protein can detect antibody levels in different species (rabbits, rats and mice) after immunization with rat HEV-ORF2, and can effectively distinguish the difference in antigen-antibody interactions after immunization with an HEV-A (human HEV, hHEV) ORF2 and the HEV-C1 ORF2. HEV-C1 ORF2-targeted specific antibodies generated in serum of rats immunized with HEV-C1 ORF2 protein can effectively block specific binding of HEV-C1 ORF2 virus-like particles to rat hepatoma cells RH-35, while serum of unimmunized rats and that of rats immunized with the HEV-AORF2 protein show no blocking effect, and thus the recombinant protein is proved to be capable of being used for preparing HEV-C1 vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the homology alignment of HEV-C1 ORF2 amino acid sequences and the generation of a consensus sequence;

FIG. 2 shows the genetic relationship of HEV-C1 ORF2;

FIG. 3 shows the plasmid map of pET-21a (+) -Rat ORF2 357-595;

FIG. 4 shows the PCR product of HEV-C1 ORF2;

FIG. 5 shows the identification of the purification of HEV-C1 ORF2 with SDS-PAGE Coomassie staining, where the samples in the lanes from left to right are Marker, sample before passing through the column, sample after passing through the column, sample eluted with 10 mL of wash buffer, sample eluted with 5 mL of elution buffer, sample before dialysis, and sample after dialysis;

FIG. 6 shows the identification of denatured and non-denatured recombinant HEV-C1 ORF2 proteins using Western blot;

FIG. 7 shows that the recombinant HEV-C1 ORF2 protein can form virus-like particles according to observation with an electron microscope;

FIG. 8 shows the antibody levels of mice, New Zealand white rabbits and rats immunized with HEV-C1 ORF2, where A, B and C are mice, New Zealand white rabbits and rats, respectively;

FIG. 9 shows the results of the specific interaction of antibody generated by immunization with recombinant HEV-C1 ORF2 protein with HEV-C1 ORF2, HEV-A ORF2 and HEV-A ORF3, where A is rat 1 and B is rat 2;

FIG. 10 shows the analysis of the interaction between serum from a HEV infected patient and HEV-A ORF2 and HEV-C1 ORF2;

FIG. 11 shows the results of the detection of HEV-C1 ORF2 protein using the polyclonal antibody prepared by immunization of a rabbit with HEV-C1 ORF2 protein;

FIG. 12 shows that HEV-C1 ORF2 can form virus-like particles, which can specifically bind to cell membrane of rat hepatoma cell RH-35 and can fully simulate the process of adsorption of rat HEV virus on the surface of susceptible cells, where A shows the binding of the control to the surface of rat hepatoma cell RH-35, and B shows the binding of HEV-C1 ORF2 to the surface of rat hepatoma cell RH-35;

FIG. 13 shows that the HEV-C1 ORF2-targeted specific antibody generated in the serum of a rat immunized with the HEV-C1 ORF2 protein can effectively block the specific binding of HEV-C1 ORF2 virus-like particles to RH-35 cells, while the serum of unimmunized rat and the serum of a rat immunized with HEV-A ORF2 protein have no blocking effect, where A is the immunofluorescence patterns of the negative control groups in which RH-35 cells are not incubated with rHEV-ORF2, and only anti-hHEV-ORF2 and rHEV-ORF2 are added, and B is the immunofluorescence patterns showing the interaction of the RH-35 cells with the rHEV-ORF2, the rHEV-ORF2 and the anti-hHEV-ORF2, and the rHEV-ORF2 and the anti-rHEV-ORF2, respectively.

DETAILED DESCRIPTION

The present invention provides a recombinant rat HEV-C1 ORF2 protein, wherein an amino acid sequence of the recombinant protein is set forth in SEQ ID No. 1, which is specifically as follows:

The recombinant HEV-C1 ORF2 protein provided by the present invention consists of 240 amino acids and has a molecular weight of 26 kDa.

In the present invention, the amino acid sequence of the recombinant HEV-C1 ORF2 protein is a consensus sequence obtained by aligning with the amino acid sequence of HEV-C1 ORF2 in NCBI database and constructing based on functional analysis of Vector NTI Advance 11.5AlignX.

The present invention also provides a recombinant rat HEV-C1 ORF2 protein, wherein the amino acid sequence of the recombinant protein has at least 90%identity to the sequence set forth in SEQ ID No. 1, and preferably has at least 95%identity. In the present invention, the sequence set forth in SEQ ID No. 1 is a consensus sequence constructed based on artificial analysis by utilizing the characteristic of consensus sequences, so that a sequence having at least 90%identity to the sequence set forth in SEQ ID No. 1 is equivalent to the sequence set forth in SEQ ID No. 1 in terms of performance and is able to obtain test effect equivalent to that of the sequence set forth in SEQ ID No. 1.

Sequence identity is typically measured in percentage identity (or similarity or homology) ; the higher the percentage, the more similar the two sequences. Methods for aligning and comparing sequences are well known in the art. Various programs and alignment algorithms are described below: Smith T, Waterman M. Comparison of biosequences. Advances in Applied Mathematics. 1981 Dec, 2 (4) : 482-489; Needleman S, Wunsch CA general method applicable to the search for similarities in the amino acid sequence of two proteins, J Mol Biol. 1970 Mar; 48 (3) : 443-53; Pearson W, Lipman D. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr; 85 (8) : 2444-8; Higgins D, Sharp P. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15; 73 (1) : 237-44; Higgins D, Sharp P. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr; 5 (2) : 151-3; Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988 Nov 25; 16 (22) : 10881-10890; Huang X, Miller W, Schwartz S, Hardison R. Parallelization of a local similarity algorithm. Comput Appl Biosci. 1992 Apr; 8 (2) : 155-65; Pearson W. Using the FASTA program to search protein and DNA sequence databases. Methods Mol Biol. 1994; 24: 307-31; Altschul S, Boguski M, Gish W, Wootton J. Issues in searching molecular sequence databases. Nat Genet. 1994 Feb; 6 (2) : 119-29. The literature above provided detailed consideration about methods for sequence alignment and calculation of homology.

For example, the alignment tool ALIGN (Myers E and Miller W. Optimal Alignments in Linear Space. CABIOS 4, 1988, 11-17) or LFASTA (Pearson W, Lipman D. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr; 85 (8) : 2444-8) can be used for sequence comparison (Internet 1996, W.R. Pearson and the University of Virginia, fasta version 2.0, released in Dec. 1996) . ALIGN compares entire sequences, while LFASTA compares local regions for similarity. For example, these alignment tools and their respective tutorials are available from the website of the National Center for Supercomputer Applications (NCSA) on the Internet. Alternatively, to compare amino acid sequences with greater than about 30 amino acids, the Blast 2 sequence function can be performed using the default BLOSUM62 matrix set to default parameters (gap existence penalty of 11, and per residue gap penalty of 1) . When short peptides (less than about 30 amino acids) are aligned, the alignment should be performed using Blast 2 sequence function, employing PAM30 matrix set to default parameters (gap opening penalty of 9, and gap extension penalty of 1) . BLAST sequence comparison systems are available, for example, from the NCBI website. (Altschul S, Gish W, Miller W, Myers E, Lipman D. Basic local alignment search tool. JMol Biol. 1990 Oct 5; 215 (3) : 403-10; Gish W, States D. Identification of protein coding regions by database similarity search. Nat Genet. 1993 Mar; 3 (3) : 266-72; Madden T, Tatusov R, Zhang J. Applications of network BLAST server. Methods Enzymol. 1996; 266: 131-41; Altschul S, Madden T,

A, Zhang J, Zhang Z, Miller W, Lipman D. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1; 25 (17) : 3389-402; Zhang J, Madden T. PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res. 1997 Jun; 7 (6) : 649-56. )

The present invention provides a gene encoding the recombinant rat HEV-C1 ORF2 protein, wherein a nucleotide sequence of the gene is set forth in SEQ ID No. 2, which is specifically as follows:

The present invention also provides an expression vector of a recombinant rat HEV-C1 ORF2 protein comprising a plasmid backbone and the gene. There is no particular limitation for the specific type of the plasmid backbone in the present invention, and conventional commercially available expression vectors, such as expression plasmids including the eukaryotic expression plasmid pcDNA3.1 and the like and related expression plasmids of slow virus/adenovirus vectors (such as pWPI and pAAV) , can be used. In a specific implementation process of the present invention, the plasmid backbone is preferably pET-21a (+) . In the present invention, the gene is preferably inserted between restriction enzyme digestion sites Nde I and Xho I of the plasmid backbone. There is no particular limitation for the method for constructing the expression vector of the recombinant rat HEV-C1 ORF2 protein, and a conventional method for constructing a recombinant vector in the art can be used. In the specific implementation process of the present invention, the recombinant expression vector is constructed by using double restriction enzyme digestion and ligation.

The present invention also provides a recombinant bacterial strain expressing the recombinant rat HEV-C1 ORF2 protein, wherein the recombinant bacterial strain is transformed with the expression vector.

In the present invention, preferably E. coli BL21 is used as host bacteria for the recombinant bacterial strain; there is no particular limitation for the method for the transformation in the present invention, and a conventional method for transformation of an expression vector in the art can be used.

The present invention provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a vaccine for preventing human infection of rat HEV. In the present invention, the vaccine is preferably a subunit vaccine. In the present invention, the vaccine comprises the recombinant rat HEV-C1 ORF2 protein and Freund’s complete adjuvant; a concentration of the recombinant rat HEV-C1 ORF2 protein in the vaccine is preferably 0.8–1.2 mg/mL, and more preferably 1 mg/mL. In the present invention, antibodies with relatively high titer can be obtained after immunization of rats, mice and New Zealand white rabbits with the vaccine.

The present invention also provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a reagent for detecting human infection of rat HEV. The present invention has found, through researches, that the antibody generated in HEV-A infected patients specifically interacts with HEV-A ORF2, but not with the recombinant rat HEV-C1 ORF2 protein. The recombinant rat HEV-C1 ORF2 protein cannot detect HEV-A infected patients, and the recombinant rat HEV-C1 ORF2 protein can be used for establishing an ELISA method to realize specific diagnosis of human infection of HEV-C1 virus.

The present invention provides a monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein. There is no particular limitation for the method for separating and purifying the monoclonal antibody and/or the polyclonal antibody, and a conventional method for separating and purifying a monoclonal antibody and/or a polyclonal antibody in the art can be used.

The present invention also provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a reagent for detecting rat HEV.

The present invention also provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a vaccine for preventing human infection of rat HEV.

The technical schemes provided by the present invention are described in detail below with reference to examples; however, the examples should not be construed as limiting the scope of the present invention.

Example 1

Alignment of HEV-C1 ORF2 Amino Acid Sequences and Design of ORF2 Consensus Amino Acid Sequence

Sequences of HEV-C1 ORF2 were downloaded from NCBI and sequence numbers were: HEV-C1HEV (AB847305) , HEV-C1 HEV (LC145325) , HEV-C1 HEV (AB847306) , HEV-C1 HEV (AB847309) , HEV-C1 HEV (AB847308) , HEV-C1 HEV(GU345042) , HEV-C1 HEV (GU345043) , HEV-C1 HEV (JN167537) , HEV-C1 HEV (KM516906) , HEV-C1 HEV (JN167538) , HEV-C1 HEV (MK050105) , HEV-C1 HEV (LC225389) , HEV-C1 HEV (AB847307) , HEV-C1 HEV (JX120573) , HEV-C1 HEV (MG813927) and HEV-C1 HEV (LC225388) .

All the downloaded sequences were imported into Vector NTI (11.5.3) , aligned using Alignment function (the results are shown in FIG. 1) and subjected to genetic relationship analysis using Guide Tree function (the results are shown in FIG. 2) , and a consensus sequence SEQ ID No. 1 of ORF2 was constructed by Consensus Calculation analysis.

SEQ ID No. 1:

Example 2

Construction of Expression Plasmid of HEV-C1 ORF2

A nucleotide sequence was designed according to the consensus amino acid sequence obtained by alignment of ORF2 sequences, codon optimization was carried out according to the host E. coli, and a gene encoding the ORF2 sequence was obtained through gene synthesis. The gene was amplified by PCR, restriction enzyme digestion sites NdeI and XhoI were added at two ends of a primer, respectively, the gene encoding the truncated ORF2 (357-595aa) fragment and a pET21a vector were subjected to double restriction enzyme digestion with NdeI and XhoI and gel extraction, and then were ligated using a T4 ligase to construct a recombinant expression plasmid pET-21a (+) -Rat ORF2357-595 (the plasmid map is shown in FIG. 3) . The recombinant expression plasmid was provided with an initiation codon and a His tag, so that the protein of interest and the His tag were expressed in a recombinant mode.

Specific steps are as follows.

A gene of the HEV-C1 ORF2 (357-595aa) consensus sequence was artificially synthesized. The length of the gene fragment was 720 bp, the gene sequence was set forth in SEQ ID No. 2, the fragment was synthesized by Suzhou Genewiz Biological Technology Co., Ltd., and specific steps are as follows:

ORF2 PCR primers were used for amplification, the length of the gene fragment was 730 bp, restriction enzyme digestion sites NdeI and XhoI were added at the 5’ end and the 3’ end, respectively, and the PCR amplification primers are as follows:

Forward primer HEV-C1 ORF2-F:

5’-ggaattccatatgattgtgcaagtgctgtttaac-3’ (SEQ ID No. 3, Tm=61℃) .

Reverse primer HEV-C1 ORF2-R:

5’-ccgctcgaggcttggtccaatggcacccag-3’ (SEQ ID No. 4) .

The PCR reaction system is shown in Table 1.

Table 1. PCR reaction system

Composition	25 μL of system
5X Q5 Reaction Buffer	5 μL
10 mM dNTPs	0.5 μL
10 μM Forward Primer	1.25 μL
10 μM Reverse Primer	1.25 μL
Template DNA
	1 ng
Q5 High-Fidelity DNA Polymerase	0.25 μL
Nuclease-Free Water	to 25 μL

The size of the PCR product was identified by agarose electrophoresis, and the results are shown in FIG. 4, and the sequence length was as expected.

The fragment of interest and the His-tagged vector pET-21a (+) were each enzyme digested with Nde I and Xho I (see Table 2 for restriction enzyme digestion system) , and then were ligated with a T4 ligase (see Table 3 for ligation system) overnight.

Table 2. Restriction enzyme digestion of fragment of interest and vector with Nde I and Xho I

Composition	25 μL of system
DNA (PCR/plasmids)	0.5 μg
10X rCutSmart Buffer	2.5 μL (1X)
Nde I	0.5 μL (20 units)
Xho I	0.5 μL (20 units)
Nuclease-free Water	to 25 μL

(1) The ratio of the amount of the fragment of interest (730 bp) to the amount of the vector fragment was 5: 1, and thus the amount of the fragment of interest was calculated (see Table 3) .

(2) The sample was placed in a ligation device at 16℃ and ligated overnight.

Table 3. Ligation system

Components	Volume
T4 DNA Ligase	1 μL
10x T4 DNA Ligase Buffer	2 μL
10x Buffer
	2 μL
Fragment of interest	55 ng

Vector fragment	80 ng
Sterilized distilled water	Make up the remaining volume
Total volume	20 μL

Transformation (DH5α)

(1) 50 μL of the cells of E. coli DH5α competent cells were thawed quickly on ice, and then 10 μL of the ligation product was added; the two were mixed by gentle rotation, placed in an ice bath and stayed for 30 min.

(2) The mixture was immediately placed in a 42℃ water bath and heat shocked for 90 s without shaking to allow it to reach an accurate heat shock temperature.

(3) The mixture was then quickly transferred to an ice bath and stayed for 2 min.

(4) 650 μL of LB culture medium was added to the sample tube in a clean bench and the mixture was well mixed gently.

(5) The sample tube was placed in a shaker at 37℃ and 220 rpm for 1 h of shake culture.

(6) The mixture was centrifuged at 5000 rpm at room temperature for 1 min.

(7) 550 μL of supernatant was discarded, and about 150 μL of the substance was left, well mixed and all added to an antibiotic plate for coating, and when there was no flowing of fluid in the plate, the plate was left to stand for 10 min, and then inverted and cultured at 37℃ for 14 h. A plurality of colony clones were picked out and cultured by using LB culture medium added with ampicillin. The plasmid was extracted by using a TIANprep Mini Plasmid Kit (DP103) and sent to Suzhou Genewiz Biological Technology Co., Ltd. for sequencing identification, and the result was correct and the plasmid was named pET-HEV-C1-ORF2-His.

Example 3

Expression and Purification of Recombinant HEV-C1 ORF2 Protein

The recombinant expression plasmid pET-21a (+) -Rat ORF2 357-595 was transformed into E. coli expression bacteria BL21, isopropyl-β-D-thiopyran and galactoside were added to induce expression, and finally bacterial liquid was collected, lysed by ultrasound, centrifuged and purified, and the expression of protein was determined by SDS-PAGE and Western blot. Specific steps are as follows:

(1) The bacterial cells were collected in 50 mL centrifuge tubes (centrifuging at 4000 rpm for 10 min to collect the precipitate) . After centrifugation for several times, the precipitate of bacterial cells was collected in a 50 mL centrifuge tube. The tubes were cryopreserved at-20℃.

(2) The cryopreserved bacterial liquid was taken out, and each tube was added with 5 mL of PBS, 50 μL of protease inhibitor and 50 μL of lysozyme (the final concentration of lysozyme was 1 mg/mL) . The mixture was well mixed and left to stand for 30 min.

(3) Ultrasonic treatment: the mixture was transferred into a 10 mL centrifuge tube and sonicated for 45 min.

(4) Centrifuging: the mixture was centrifuged at 11000 rpm for 30 min at 4℃, and the supernatant was discarded.

(5) The residue was homogenized and washed three times with 5 mL of inclusion body washing solution and then centrifuged at 12000 rpm for 10 min at 4℃ to collect the perticipate.

(6) The precipitate was dissolved in 5 mL of inclusion body denaturation solution (Table 4) and centrifuged at 12000 rpm for 10 min at 4℃ to collect the supernatant.

(7) The supernatant was diluted by using 5 mL of inclusion body renaturation solution (Table 5) .

Table 4. Formula of inclusion body denaturing solution

pH adjusted to 8.0

Table 5. Preparation of inclusion body renaturation solution

pH adjusted to 8.0

(8) The storage buffer was removed by washing with 4 mL of deionized water, and the column was equilibrated using at least 5 mL of lysis buffer.

(9) The protein sample was loaded into the column to allow the protein to be adsorbed onto the column.

(10) The column was washed with 15 mL of wash buffer.

(11) Eluting with elution buffer: the protein of interest was washed with 5 mL of elution buffer, and the eluted protein was collected with an EP tube.

(12) The purification efficiency was identified by Coomassie blue staining using SDS-PAGE. The operation steps are as follows:

①Extraction of total protein: a corresponding amount of 5×protein loading buffer was added, and the mixture was mixed well, boiled for 10 min and cooled, and then the sample was stored at-20℃.

②SDS-PAGE electrophoresis: 12.5%separation gel and 4%concentration gel were prepared. Firstly, the separation gel was prepared, mixed well and then filled into the gel plate, absolute ethanol was used for levelling the gel surface, and 40 min later, the absolute ethanol on the upper layer was poured out, and residual liquid was adsorbed with absorbent paper; then the concentration gel was prepared, mixed well and then filled into the upper layer, and after a comb was inserted, the gel was left to stand for about 40 min at room temperature. After the gel was fully solidified, it was put into a refrigerator at 4℃ and stored for later use. See Table 6 for preparation of electrophoresis buffer. The gel plate was put into an electrophoresis frame, and the electrophoresis buffer was added and then the comb was removed. 15 μL of protein sample was added into each gel hole except for one, which was added with a protein Marker. The electrophoresis frame was put into an electrophoresis tank, and the electrophoresis was started after the electrophoresis buffer in the inner tank and the outer tank was replenished. Line pressing was performed for 30 min at 70 V, and then the voltage was changed to 120 V when the sample was electrophoresed to the boundary line between the concentration gel and the separation gel, and the electrophoresis was stopped when bromophenol blue moved to the bottom of the separation gel.

Table 6. Formula of electrophoresis buffer

The results of Coomassie blue stained SDS-PAGE are shown in FIG. 5. ORF2 was expressed and bound well to the Ni-NTA column and was able to be eluted normally by the elution buffer. Western blot results are shown in FIG. 6. Non-denatured ORF2 formed a dimer, which changed to a monomer after denaturation of the protein.

(12) Western blot was performed using a His-tagged antibody (1: 5000 dilution) and ORF2 protein was determined for denaturation and non-denaturation, and the operation steps are as follows.

Protein extraction and SDS-PAGE are shown as in the step (11) , and on this basis, a PVDF membrane with the same size as the gel block of interest was obtained by cutting, soaked in methanol for 30 s to be fully activated, and then placed in recycled wet transfer buffer to be soaked for 3–5 min. A white filter pad with the same size as the gel block was obtained by cutting, fully soaked, and then put on a wet transfer clamp, and the membrane transfer was performed in an electroporator for wet transfer. The sequence was as follows: white plate-sponge pad-white filter paper-PVDF membrane-gel-white filter paper-sponge pad-black plate. The power was connected and the voltage was stabilized at 100 V, and wet transfer was performed for 100 min. The membrane was soaked in a blocking solution (5%skimmed milk powder) and put in a shaker for 2 h of blocking at room temperature. After the blocking was finished, the PVDF membrane was cleaned simply, cut to be consistent with the protein of interest in terms of size and placed into an antibody incubation box. The His antibody serum was subjected to 5000-fold dilution by using 1%BSA and then added into the incubation box. The mixture was incubated on a shaker at 4℃ overnight. The antibody incubation box was taken out the next day. After rewarming on the shaker for 30 min at room temperature, the primary antibody was pipetted out and washed 3 times (10 min each time) with TBST wash buffer in a shaking manner. The PVDF membrane was placed in the antibody incubation box, and the diluted anti-rabbit IgG antibody was added (the dilution ratio of 1: 5000, the diluent being 1%BSA) . The mixture was incubated for 2 h on a shaker at room temperature, and then the secondary antibody was pipetted out and washed 3 times (10 min each time) with TBST wash buffer in a shaking manner. ECL was used for chromogenic reaction in Western blot.

Table 7. Preparation of lysis buffer

pH adjusted to 8.0

Table 8. Preparation of wash buffer

pH adjusted to 8.0

Table 9. Preparation of elution buffer

pH adjusted to 8.0

Example 4

Recombinant HEV-C1 ORF2 protein forms virus-like particles capable of simulating the structure of HEV-C1 virus and having strong immunogenicity and is a candidate strain of HEV-C1 subunit vaccine

100 μL (1 drop) of stock solution of 2 mg/mL recombinant HEV-C1 ORF2 protein solution was added dropwise on a copper net provided with a supporting film and left to stand for 2 min, and excess liquid was removed from the edge of the copper net by using absorbent paper. 100 μL (1 drop) of 1.5%uranium acetate (pH=7) was then added dropwise, and the mixture was left to stand for 2 min, and excess liquid was removed from the edge of the copper net by using absorbent paper. The mixture was air dried for 2 min and then observed by using an electron microscope.

The observation results are shown in FIG. 7. It was found that the recombinant HEV-C1 ORF2 protein was able to form virus-like particles with the size of 40–60 nm capable of simulating the capsid structure of HEV-C1 virions and having the immunogenicity equivalent to the virions, and was a candidate strain of the HEV-C1 subunit vaccine.

Example 5

Establishment of Serum ELISA Method with Recombinant HEV-C1 ORF2 Protein, and Determination of Serum Antibody Titer of Animal Immunized with the Protein

1. Immunizing rats, mice and New Zealand white rabbits with the recombinant HEV-C1 ORF2 protein, and obtaining serum at different time periods

1.1 Immunization of rats: 500 μL of rat blood was obtained from the inner canthus using capillary pipette before immunization, and serum was separated to be used as a negative control. The purified recombinant HEV-C1 ORF2 protein was diluted to 100 μL (200 μg) and emulsified with equal volume of Freund’s complete adjuvant, and after alcohol was sprayed at multiple sites on the back of the rat for sterilization, subcutaneous injection was performed for immunization. Two weeks after immunization, the rat was again anesthetized, and 500 μL of serum after the first immunization was obtained and detected for its titer. Two weeks later, the second immunization was performed. In this experiment, the rat needed to be immunized six times. Two weeks after the final immunization, all the blood of the rat was obtained from the heart and the rat was sacrificed, and the serum titer after the sixth immunization was detected.

1.2 Immunization of mice: 200 μL of mouse blood was obtained from the inner canthus using capillary pipette before immunization, and serum was separated to be used as a negative control. The purified recombinant HEV-C1 ORF2 protein was diluted to 50 μL (100 μg) and emulsified with equal volume of Freund’s complete adjuvant, and subcutaneous injection was performed for immunization. One week after the immunization, blood was taken from the inner canthus of the mouse to obtain 200 μL of mouse serum after the first immunization, and the serum titer was detected. Two weeks after the immunization, the second immunization was performed, and two weeks after the second immunization, the third immunization was performed. In this experiment, the mouse needed to be immunized six times. One week after the final immunization, all the blood of the rat was obtained from the eyeball and the rat was sacrificed by cervical dislocation, and the serum titer after the sixth immunization was detected.

1.3 Immunization of New Zealand white rabbits: 2 mL of blood was obtained from the ear vein of the rabbit before injection for immunization, and serum was separated to be used as a negative control. The recombinant purified HEV-C1 ORF2 protein (1 mg) was diluted to 1 mL with normal saline and emulsified with 1 mL of Freund’s complete adjuvant in equal volume for immunization, and the second immunization was performed 2 weeks later. In this experiment, the injection for immunization needed to be performed 5 times, and before each immunization, the blood needed to be obtained from the ear vein to detect the titer of the antibody generated after the last immunization. One week after the final immunization, the blood was obtained from the ear vein to detect the titer, and then the rabbit was anesthetized with 10 mL of 10%chloral hydrate, and all the blood of the rabbit was obtained from the heart.

2. Establishing an ELISA method and determining the antibody level of anti-ORF2 in different kinds of animals

① An ELISA plate was coated with 200 ng of the recombinant HEV-C1 ORF2 protein, and another plate was coated with ORF3, also with His-tag, as a negative control;

② The obtained serum of animals immunized with recombinant HEV-C1 ORF2 protein was subjected to transverse fold dilution from 1: 1000, and then the diluted serum was incubated with the recombinant HEV-C1 ORF2 protein on the ELISA plate for 1 h at 37℃. The incubation serum was then discarded, and the plate was washed with PBST three times;

③ IgG-HRP secondary antibodies (1: 500 dilution) against different animal sera (anti-rat, anti-mouse and anti-rabbit) were each added, incubated for 1 h at 37℃ and then discarded, and the plate was washed with PBST three times;

④ TMB color developing solution was added for color developing for 2 min, H ₂SO ₄ was added to stop the reaction, and an ELISA spectrophotometer plate reader was used for detecting the results at450 nm.

The reagents used were:

① Coating solution: pH 9.6, 0.05 M carbonate buffer

② Stopping solution: 1 N sulfuric acid

③ Blocking solution: 5% (v/v) milk

④ ELISA washing solution: PBS+0.05%Tween20.

The detection results showed that after the mouse was immunized with the recombinant HEV-C1 ORF2 protein for 6 times, the antibody titer reached a very high level, and the antibody still had very high antibody titer after being subjected to 1000-fold to 32000-fold dilution (A in FIG. 8) ; after the New Zealand white rabbit was immunized, its serum still stayed in the platform stage of ELISA reaction after 16000-fold dilution, and it could still have good interaction with the recombinant HEV-C1 ORF2 protein after 128000-fold dilution (B in FIG. 8) ; after 5 immunizations of the mouse, its serum was still able to interact with the ORF2 protein after 4000-fold dilution.

The results above showed that the ELISA method featuring coating with the antigen can detect HEV-C1 ORF2 serum antibody of different species after immunization, and the results show antibody concentration dependence, so that the ELISA method can be established by using the recombinant HEV-C1 ORF2 protein as the antigen for diagnosing HEV-C1 infected patients, and the immune experiments of different animals above show that the HEV-C1 ORF2 protein has very good immunogenicity. Example 6

Cross Immunity of Antibody Generated in Rat Immunized with Recombinant HEV-C1 ORF2 Protein to HEV-C1 ORF2 and HEV-A ORF2

The recombinant HEV-C1 ORF2 protein and proteins of HEV-A ORF2 and HEV-A ORF3 were each used to coat an ELISA plate, and the serum of the rat immunized with the recombinant HEV-C1 ORF2 protein five times was subjected to fold dilution, and the diluted serum was incubated with the three different antigens. The method is as follows:

(1) ELISA plates were coated with 200 ng of antigen proteins (HEV-C1 ORF2, HEV-A ORF2 and HEV-A ORF3) , respectively;

(2) The serum obtained from two rats immunized with the recombinant HEV-C1 ORF2 protein was subjected to transverse fold dilution from 1: 1000, and then the diluted serum was incubated with each of the antigen proteins on ELISA plates at 37℃ for 1 h; the incubation serum was then discarded, and the plate was washed with PBST three times;

(3) Anti-rat IgG-HRP secondary antibody (1: 500 dilution) was added, and the mixture was incubated at 37℃ for 1 h; the secondary antibody was then discarded, and the plate was washed with PBST three times;

(4) TMB color developing solution was added for color developing for 2 min, H ₂SO ₄ was added to stop the reaction, and an ELISA spectrophotometer plate reader was used for detecting the results at 450 nm.

The serum titers of two immunized rats were determined, and it was found that the antibody generated after the immunization with the recombinant HEV-C1 ORF2 protein could specifically interact with HEV-C1 ORF2, but could not interact with the proteins of HEV-A ORF2 and HEV-A ORF3. The results above show that the level of the cross-immune reaction between the HEV-A ORF2 protein and the HEV-C1 immune antibody is very low, so that the ELISA detection method established with the HEV-A ORF2 cannot be used for diagnosing rat HEV infection, and therefore, a diagnostic kit established with the HEV-C1 is very important.

Example 7

Detection of Cross-Immune Reaction Between Serum of HEV-A Virus Infected Patient and Recombinant HEV-C1 ORF2 Protein

The recombinant HEV-C1 ORF2 protein and the protein of HEV-A ORF2 were each used to coat an ELISA plate, and the serum of an HEV-A infected patient was subjected to fold dilution, and the diluted serum was incubated with the two different antigens. The experimental process is as follows:

(1) ELISA plates were coated with 200 ng of recombinant HEV-C1 ORF2 protein and HEV-A ORF2, respectively;

(2) The serum of HEV-A patient was subjected to fold dilution from 1: 100, and 100 μL of the diluted antibody was added and incubated with the antigen protein on the ELISA plate at 37℃ for 1 h; the incubation serum was then discarded, and the plate was washed with PBST three times;

(3) Anti-rat IgG-HRP secondary antibody (1: 1000 dilution) was added, and the mixture was incubated at 37℃ for 1 h; the secondary antibody was then discarded, and the plate was washed with PBST three times;

The results were identified and it was found that the antibody generated in the HEV-A infected patient specifically interacted with the HEV-A ORF2, but not with the recombinant HEV-C1 ORF2 protein. The experiment proves that the recombinant HEV-C1 ORF2 protein cannot be used to detect HEV-A infected patients, and the protein can be used to establish an ELISA method for diagnosing human infection of HEV-C1 virus.

Example 8

The polyclonal antibody generated due to immunization with the recombinant HEV-C1 ORF2 protein can be used as a means for experimental detection of HEV-C1.

The length of the ORF2 gene sequence was 730 bp. The rHEV strain LCK-3110 (GenBank: MG813927.1) was used as a template, the pcDNA3.1-Flag was used as a vector, and restriction enzyme digestion sites Xho I and EcoRI were added at the upper stream and the down stream, respectively, to design a primer for amplifying the gene sequence.

Upstream primer: 5’-aacctcgagatgtctgtcgttgtcgtgctcg-3’ (SEQ ID No. 5)

Downstream primer: 5’-aacgaattcgacactgtcggctgctacggct-3’ (SEQ ID No. 6)

PCR amplification of the full-length ORF2 fragment

(1) The following reaction mixture was added to a 0.2 mL EP tube, and the reaction system is shown in Table 10.

Table 10. PCR reaction system

Components	Volume
Primer STAR Max DNA polymerase	25 μL
Forward Primer (10 μM)	1 μL
Reverse Primer (10 μM)	1 μL
Template
	2 μL
Sterilized distilled water	21 μL
Total volume	50 μL

(2) The sample tube was placed in a PCR instrument and the reaction procedure is shown in Table 11 (number of cycles: 35) . Thus, the PCR product of ORF2 was obtained.

Table 11. Procedure of PCR amplification reaction

Temperature	Time
Pre-denaturation at98℃	5 min
Denaturation at98℃	10 s
Annealing at65℃	30 s
Extension at72℃	40 s

Gel extraction of PCR product

(1) After the PCR product was electrophoresed with 2%agarose gel, the gel block of interest was carefully cut off under an ultraviolet lamp.

(2) The empty 1.5 mL EP tube was weighed after high pressure treatment, and the weight was recorded; the gel block of interest was put into the EP tube and the tube was weighed again; the difference of the two weight values was the weight of the gel block of interest.

(3) The correct amount of binding buffer was added to the EP tube according to the rule of 1 μg of gel+1 μL of binding buffer.

(4) The tube was placed in a 55℃ water bath and then taken out 10 min later; the EP tube was carefully observed to see whether the gel block was completely dissolved, and if not, the tube could be heated for a short time again until the gel block was completely dissolved.

(5) The DNA agarose solution was transferred to an adsorption column, left to stand for 2 min, and centrifuged at 12000 rpm for 75 s. (the adsorption column could only accommodate 700 μL of solution each time, and if the solution could not be added into the adsorption column one time, the rest part could be added into the adsorption column after centrifugation)

(6) Waste liquid was discarded, and 700 μL of washing buffer was added into the adsorption column and centrifuged at 10000 rpm for 75 s.

(7) The previous step was repeated.

(8) Waste liquid was discarded, and centrifugation at 10000 rpm was performed for 2 min without adding another material.

(9) The column was transferred to a new, high pressure-treated 1.5 mL EP tube, the lid opened, and the tube was left to stand for 2 min.

(10) 30 μL of elution buffer was added to the center of the column, and after the tube was left to stand for 2 min, the mixture was centrifuged at 13000 rpm for 1 min.

(11) The concentration of the extracted product was determined and recorded on the wall of the EP tube, and the tube was stored at-20℃.

Restriction enzyme digestion of the fragment of interest and the vector

(1) The following reaction mixture was added to a 0.2 mL EP tube.

Table 12. Restriction enzyme digestion system

Components	Volume
Enzyme
1	1 μL
Enzyme
2	1 μL
10x Buffer
	2 μL
DNA	≤1 μg
Sterilized distilled water	Make up the remaining volume
Total volume	20 μL

(2) After the sample was mixed well according to the reaction system, the sample was centrifuged for a short time, and then the sample was placed in an incubator at 37℃for 3 h of restriction enzyme digestion.

Ligation of the fragment of interest to the vector fragment

(1) The ratio of the amount of the fragment of interest (730 bp) to the amount of the vector fragment (5500 bp) was 3: 1, and thus the amount of the fragment of interest was calculated (see Table 13) .

(2) The sample was placed in a ligation device at 16℃ and ligated overnight.

Table 13. Ligation system

Components	Volume
T4 DNA Ligase	1 μL
10x T4 DNA Ligase Buffer	2 μL
10x Buffer
	2 μL
Fragment of interest	32 ng
Vector fragment	80 ng
Sterilized distilled water	Make up the remaining volume
Total volume	20 μL

Transformation of E. coli (DH5α)

(1) 50 μL of the cells of E. coli DH5αcompetent cells were thawed quickly on ice, and then 10 μL of the ligation product was added; the two were mixed by gentle rotation, placed in an ice bath and stayed for 30 min.

(6) The mixture was centrifuged at 5000 rpm at room temperature for 1 min.

(7) 550 μL of supernatant was discarded, and about 150 μL of the substance was left, well mixed and all added to an antibiotic plate for coating, and when there was no flowing of fluid in the plate, the plate was left to stand for 10 min, and then inverted and cultured at 37℃ for 14 h.

The recombinant plasmid was sent for sequencing identification, and the result was correct and the plasmid was pcDNA3.1-ORF2-FLAG.

2. Detection of ORF2 protein eukaryotic expression positive cells with polyclonal antibody generated by recombinant HEV-C1 ORF2 protein

HEK 293T cells were transfected with the pcDNA3.1-ORF2-flag plasmid constructed above.

(1) The cells were seeded into a 6-well plate at 6×10 ⁵ per well, and 2 mL of DMEM high-sugar culture medium containing 10%fetal bovine serum was added, and after being well mixed, the mixture was incubated in an incubator at 37℃ and 5%CO ₂ for culture, and 124 h later, the cell confluence reached 80%.

(2) 4 μg of plasmid and 12 μL of liposome PEI were each dissolved in 100 μL DMEM high-sugar culture medium and left to stand at room temperature for 5 min, and then the plasmid solution was added into the liposome solution, and the two were well mixed gently and left to stand at room temperature for 20 min.

(3) The DNA-liposome mixture was added into the six-well plate at 200 μL per well, and then the mixture was well mixed and incubated in an incubator at 37℃ and 5%CO ₂, and the medium was changed into a DMEM high-sugar culture medium containing 10%fetal bovine serum 5 h later, thus completing cell transfection.

(4) 4%paraformaldehyde was added to immobilize the cells, and the mixture was left to stand at room temperature for 30 min.

(5) The paraformaldehyde was discarded, PBS was added along the side wall, and the plate was washed 2 times to remove the residual paraformaldehyde.

(6) 0.2%Triton was added for perforation until the cells were all covered, and the mixture was left to stand at room temperature for 15 min.

(7) Triton was discarded, and the plate was washed twice with PBS added along the side wall.

(8) Blocking solution (5%skimmed milk powder) was added to the wells, and the mixture was left to stand at room temperature for 60 min.

(9) Polyclonal antibody (rabbit antibody) generated by recombinant HEV-C1 ORF2 protein was diluted at 1: 2500 and Flag antibody (mouse antibody) was diluted at 1: 5000, and they were incubated together at 37℃ for 1 h.

(10) PBS was added along the side wall, and the plate was placed on a shaker and shaken slowly, and then the plate was washed 3 times (5 min each time) .

(11) Cells were stained with Cora Lite 488 (anti-rabbit) /594 (anti-mouse) conjugated IgG (green/red) (1: 500 dilution) and Hochest (blue) (1: 500 dilution) in the dark and incubated at 37℃ for 2 h.

(12) PBS was added along the side wall, and the plate was placed on a shaker and shaken slowly, and then the plate was washed 3 times (5 min each time) .

(13) The cells were observed and photographed under an inverted fluorescence microscope.

(14) The results are shown in FIG. 11: in the immunofluorescence experiment, the rabbit anti-rHEV-ORF2 polyclonal antibody and the mouse anti-Flag antibody could specifically detect the Flag-tag-containing rHEV-ORF2 protein.

Example 9

Polyclonal antibody generated by immunization with recombinant HEV-C1 ORF2 protein can block the binding of virus-like particles to the cell surface, and the recombinant protein can be used as the main component of HEV-C1 subunit vaccine.

1. The adsorption of HEV-C1 ORF2 recombinant virus-like particles on the surface of a RH-35 cell was carried out according to the following experimental procedure:

(1) RH-35 rat hepatoma cells (purchased from Procell Life Science&Technology Co., Ltd. ) were plated on a 12-well plate at a density of 2×10 ⁵ cells/well and incubated overnight in an incubator at 37℃ and 5%CO ₂ saturation.

(2) The next day, the cell density reached 80%; HEV-C1 ORF2 purified protein (afinal concentration of 20 μg/mL per well) was mixed with PBS, and a total of 300 μL of the two was mixed in a 1.5 mL EP tube, and the control group was PBS; the tubes were incubated at 37℃ for 1 h.

(3) The cell supernatant in the plate was discarded, the cells were washed twice with pre-cooled PBS, 300 μL of the protein and PBS incubation mixture was added to each well, 300 μL of the incubation PBS was added to control wells, the plate was placed on ice and incubated on a shaker gently for 30 min.

(4) The liquid in the plate was discarded, the cells were washed with pre-cooled PBS, and the plate was placed on ice and gently washed 3 times (5 min each time) on a shaker.

(5) 300 μL of 2%paraformaldehyde (4%paraformaldehyde diluted to 2%in PBS) was added to each well, and the plate was placed on ice and incubated gently on a shaker for 30 min for immobilization of cells.

(6) Step (4) was repeated.

(7) 1%Casein (prepared with PBST) was added at 300 μL per well as a blocking solution, and the cells were blocked at room temperature for 1 h.

(8) The blocking solution was discarded, and the cells were washed with PBS twice on a shaker (5 min each time) .

(9) HEV-C1 ORF2 rabbit polyclonal antibody immune serum was added, diluted with 1%BSA at 1: 3000 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.

(10) The rabbit fluorescent secondary antibody and Hoechst were diluted with 1%BSA at 1: 500 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.

(11) The binding of the cells to the protein was observed and photographed under an inverted fluorescence microscope, and the results were analyzed.

The results are shown in FIG. 12, and they suggest that virus-like particles formed by recombinant HEV-C1 ORF2 protein can specifically bind to the cell surface of RH35, and can simulate the biological characteristics of the binding of rat HEV to surface receptor of host cells.

Competitive binding assay for rat immune serum

(1) RH-35 rat hepatoma cells were plated on a 96-well plate at a density of 1×10 ⁴ cells/well and incubated overnight in an incubator at 37℃ and 5%CO ₂ saturation.

(2) The next day, the cell density reached 80%; HEV-C1 ORF2 and HEV-A ORF2 purified proteins were mixed with rat immune serum, and there were three groups: rat negative serum diluted with PBS at a final concentration of 1: 20+20 μg/mL HEV-C1 ORF2 protein; HEV-C1 ORF2 rat immune serum diluted with PBS at a final concentration of 1: 20+20 μg/mL HEV-C1 ORF2 protein; HEV-A ORF2 rat immune serum diluted with PBS at a final concentration of 1: 20+20 μg/mL HEV-C1 ORF2 protein; the mixture was incubate for 1 h at 37℃.

(3) The cell supernatant in the plate was discarded, the cells were washed twice with pre-cooled PBS, and 100 μL of the incubation mixture of corresponding protein, rat serum and PBS was added to each well; the plate was placed on ice and incubated on a shaker gently for 30 min.

(4) The liquid in the plate was discarded, the cells were washed with pre-cooled PBS, and the plate was placed on ice and gently washed 3 times (5 min each time) on a shaker by shaking slowly.

(5) 100 μL of 4%paraformaldehyde was added to each well, and the plate was placed on ice and incubated on a shaker for 30 min by shaking slowly for immobilization of cells.

(6) Step (4) was repeated.

(7) 1%Casein (prepared with PBST) was added at 100 μL per well as a blocking solution, and the cells were blocked at room temperature for 1 h.

(9) HEV-C1 ORF2 rabbit immune serum was added, diluted with 1%BSA at 1: 3000 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.

(11) The adsorption of the HEV-C1 and the HEV-A recombinant virus-like particles on the surface of RH-35 rat hepatoma cells after the rat immune serum was added was observed under an inverted fluorescence microscope.

The experimental results are shown in FIG. 13, and they suggest that the HEV-C1 ORF2-targeted specific antibody generated in the serum of a rat immunized with the recombinant HEV-C1 ORF2 protein can effectively block the specific binding of HEV-C1 ORF2 virus-like particles to RH-35 cells, while the serum of unimmunized rat and the serum of a rat immunized with HEV-A ORF2 protein have no blocking effect. The experiment proves that the antibody with protection activity is generated in the serum of a rat immunized with the recombinant HEV-C1 ORF2 protein, and the HEV-C1 can be prevented from invading host cells. The recombinant HEV-C1 ORF2 protein can be used as an effective candidate vaccine, while the antibody generated in the serum of rats immunized with the HEV-A ORF2 protein has no cross-protection activity for the rat HEV.

The above description is only preferred embodiments of the present invention, and it should be noted that, for those of ordinary skill in the art, various improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications shall also fall within the protection scope of the present invention.

Claims

A recombinant rat HEV-C1 ORF2 protein, wherein an amino acid sequence of the recombinant protein is set forth in SEQ ID No. 1;

or the amino acid sequence of the recombinant protein has at least 90%identity to a sequence set forth in SEQ ID No. 1.
A gene encoding the recombinant rat HEV-C1 ORF2 protein according to claim 1, wherein a nucleotide sequence of the gene is set forth in SEQ ID No. 2.
An expression vector of a recombinant rat HEV-C1 ORF2 protein, comprising a plasmid backbone and the gene according to claim 2.
The expression vector of the recombinant rat HEV-C1 ORF2 protein according to claim 3, wherein the gene is inserted into a multiple cloning site of the plasmid backbone.
A recombinant bacterial strain expressing the recombinant rat HEV-C1 ORF2 protein according to claim 1, wherein the recombinant bacterial strain is transformed with the expression vector according to claim 3 or 4.
Use of the recombinant rat HEV-C1 ORF2 protein according to claim 1 in preparing a vaccine for preventing human infection of rat HEV.
Use of the recombinant rat HEV-C1 ORF2 protein according to claim 1 in preparing a reagent for detecting human infection of rat HEV.
A monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein according to claim 1.
Use of the monoclonal antibody and/or the polyclonal antibody according to claim 8 in preparing a reagent for detecting rat HEV.
Use of the monoclonal antibody and/or the polyclonal antibody according to claim 8 in preparing a vaccine for preventing human infection of rat HEV.