CN116790520A

CN116790520A - CEA protein capable of recognizing tumor surface, short chain phagemid expressing 3C protein, construction and application thereof

Info

Publication number: CN116790520A
Application number: CN202310735301.8A
Authority: CN
Inventors: 刘婷婷; 蔡良良
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2023-06-20
Filing date: 2023-06-20
Publication date: 2023-09-22

Abstract

The invention relates to a CEA protein capable of recognizing tumor surface, a short chain phagemid expressing 3C protein and construction and application thereof, belonging to the technical field of transformation of phagemid vectors and construction reasons thereof. The invention is to place the non-structural protein 3C of the Sein-card valley virus (SVV) in a eukaryotic expression frame, connect with an M13 phage packaging element, and simultaneously introduce a single-chain antibody sequence of carcinoembryonic antigen into the pIII gene of the M13 phage to prepare phagemid plasmids. After the phagemid plasmid is transformed into a host, co-transfecting helper phage M13KO7-ACEA scFv to obtain a short-chain phagemid which efficiently targets tumor cells, and identifying the short-chain phagemid by a transmission electron microscope, fluorescent observation and the like. The invention integrates phage pIII display system and eukaryotic expression element for the first time, forms short chain phagemid with targeting and gene expression in eukaryotic cells, enriches phage morphology, and provides new tools and methods for biological and medical application.

Description

CEA protein capable of recognizing tumor surface, short chain phagemid expressing 3C protein, construction and application thereof

Technical Field

The invention relates to a CEA protein capable of recognizing tumor surface, a short chain phagemid expressing 3C protein and construction and application thereof, belonging to the technical field of transformation of phagemid vectors and construction reasons thereof.

Background

Phage, which is a bacterial virus and highly evolved nanomachines, can recognize bacterial cell walls, transfer genetic information, specifically kill or remodel its host, is an existing multifunctional gene transfer material, has little side effects, and is easily cleared from the body by lysosomal degradation, causing little adverse reaction [1]. The novel oncolytic virus, serrata Valley Virus (SVV) non-structural protein 3C, can induce apoptosis, but the inherent tropism of the virus [2] and the disadvantage of high production cost thereof bring certain limitation to clinical application. The research uses M13 phage as nano delivery carrier, inserts SVV 3C gene, AAV reverse terminal repetitive sequence (ITR) and related element expression cassette participating in eukaryotic expression into M13 phage genome by molecular biology technology, and simultaneously fusion expresses single chain antibody (scFv) specifically combined with carcinoembryonic antigen (CEA) on its pIII protein by using M13 phage display technology to finally obtain short chain M13 phagemid with targeting CEA and oncolysis, and researches its oncolysis effect and oncolysis mechanism by in vivo and in vitro related experiments after polyethylene glycol (PEG) modification. The short-chain M13 phagemid constructed by the research can be rapidly positioned in tumor cells and express pro-apoptosis proteins to kill the tumor cells, and provides a novel phage treatment platform and a gene treatment idea for gene treatment of tumors.

The phagemid system can insert larger fragments and the vector is more stable than phage vectors, yet retains the multivalent display necessary for efficient cell binding and internalization. Phagemid particles rescued with helper phages had higher transduction efficiencies than full-length phages, and the specificity of multivalent phagemid targeting was comparable to that of the full-length phages from which they were derived [3]. The length of filamentous phage is determined by the size of its genome, and if viral DNA is lengthened or shortened by artificially increasing or decreasing fragments, the number of pVIII proteins and the length of the virion are proportionally increased or decreased [4]. It has been studied that Ff phage-derived nanoparticles (Ff-nano) [5,6] with a length of about 50nm are obtained by helper phage infection of circular ssDNA whose genome contains only elements (replication origin and packaging signal) necessary for Ff phage packaging, and the head pIII molecule of this short-chain phagemid fuses the polypeptide targeting the target molecule and the cell, and the relative effective collision efficiency of the target site with the target is significantly increased due to the small head, thus improving the efficiency of targeting the target [5]. The shorter length of Ff-nano compared to full length M13 phage particles can prevent filament bending and confer higher stability. Recent studies have shown that phagemid particles of different lengths can be obtained by engineering the length of the phagemid vector, and that by tail intravenous injection of short phagemid and M13 phage, the relative affinity of M13 phage to tumor tissue and tumor accumulation index is much lower than for short phagemid, suggesting that shrinking the phage length can overcome the limitations of the blood-tumor endothelial barrier [7]. The ability to regulate capsid display, surface loading, phage length, and circular single stranded DNA gene content makes recombinant M13 phage particles an ideal delivery platform.

Cancer cells must be accompanied by changes in their phenotype and genotype during growth, infiltration and metastasis, resulting in the high expression of certain tumor-associated genes and secretion into host fluids, which can produce many tumor markers. With the development of proteomics, many tumor markers have been widely used as screening tools for high risk groups in annual physical examination [8]. Carcinoembryonic antigen (carcinoembryonic antigen, CEA), also known as CEACAM5 or CD66e, is found in malignant tumors of the endothelial-derived epithelium of the gastrointestinal tract and pancreas, and is recognized as a marker of digestive tract tumors. The antigen is a glycoprotein, which consists of a single chain protein (about 800 amino acids) and sugar side chains, and exists on the surface of cancer cells differentiated from endodermal cells, and is a structural protein of cell membranes, and the C-terminal region of the antigen is anchored on the cell membranes through phosphatidylinositol [9,10]. Part of CEA is formed in the cytoplasm, is secreted outside the cell through the cell membrane, and then enters the surrounding body fluids, and can be detected in various body fluids. Carcinoembryonic antigen is used as a most common tumor marker in human medicine and is widely used as a diagnostic and monitoring index for various digestive system tumors. Carcinoembryonic antigen exists in serum of malignant tumor patients such as colon, rectum, esophagus, stomach, liver, pancreas and the like, and the content of carcinoembryonic antigen is obviously higher than that of non-tumor patients. The high expression of CEA has been demonstrated in colon, breast, lung, stomach and ovarian cancers [11], 12, 8. In experimental researches, CEA is used as a specific promoter of lung cancer or colon cancer, and has obvious curative effect on lung cancer by combining with other chemotherapeutics (such as paclitaxel). Therefore, by targeting tumor cells of CEA with high expression, the possibility is provided for eliminating cancer. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker, and can not be used as a specific index for diagnosing a certain malignant tumor, but has important clinical value in the aspects of differential diagnosis, disease monitoring, curative effect evaluation and the like of malignant tumors.

In summary, based on the phage capsid protein displaying specific antibodies targeting host cells, the following design is proposed by utilizing the characteristic that AAV cis-acting elements can be efficiently expressed in host cells:

the 5 copies of scFv specifically bound to CEA are displayed by using M13 phage pIII capsid protein to obtain recombinant phage M13KO7-ACEA scFv (ACEA scFv: scFv of anti-CEA); inserting eukaryotic expression related elements such as SVV 3C genes and AAV cis-acting elements between a phagemid pUC57-LS7 replication origin and a packaging sequence to obtain a phagemid pUC57-LS7-3C; super-infecting the helper phage M13KO7-ACEA scFv to obtain multivalent short-chain M13 phagemid M13-3C-ACEA scFv; based on the tumor targeting of enhancing the permeation retention (EPR) effect, the prepared phagemid M13-3C-ACEA scFv can reduce the nonspecific effect with cells or tissues after being modified by PEG, reduce the phagocytosis of an in vivo immune system and prolong the body circulation time; verifying targeting, stability and safety of short-chain M13 phagemid M13-3C-ACEA scFv; the targeting efficiency and oncolytic effect of long-chain phages and short-chain phagemids in vitro and in vivo colon cancer treatment were simultaneously evaluated and the mechanism thereof was elucidated.

The M13 phagemid obtained by the research shortens (reduces non-specific adsorption) through biotechnology, displays a single-chain antibody (targeted tumor) for recognizing tumor cell CEA and carries genes capable of inducing apoptosis, realizes long circulation through PEG modification, reduces non-specific adsorption and improves targeting efficiency, and further provides theoretical basis and experimental basis for developing a novel carrier delivery platform and tumor treatment.

Reference is made to:

[1]S.Y.Yoo,H.-E.Jin,D.S.Choi,M.Kobayashi,Y.Farouz,S.Wang,S.-W.Lee,M13Bacteriophage and Adeno-Associated Virus Hybrid for Novel Tissue Engineering Material with Gene Delivery Functions,Advanced healthcare materials,5(2016)88-93.

[2]R.PS,B.KD,H.LM,G.S,J.BH,I.N,H.C,L.SS,S.KL,V.AJ,Y.J,W.DN,R.CM,H.PL,Seneca Valley virus,a systemically deliverable oncolytic picornavirus,and the treatment of neuroendocrine cancers,Journal of the National Cancer Institute,99(2007)1623-1633.

[3]D.Larocca,K.Jensen-Pergakes,M.Burg,A.Baird,Receptor-targeted gene delivery using multivalent phagemid particles,Molecular therapy:the journal of the American Society of Gene Therapy,3(2001)476-484.

[4]A.A.Aksyuk,M.G.Rossmann,Bacteriophage assembly,Viruses,3(2011)172-203.

[5]S.Sattar,N.Bennett,W.Wen,J.Guthrie,L.Blackwell,J.Conway,J.Rakonjac,Ff-nano,short functionalized nanorods derived from Ff(f1,fd,or M13)filamentous bacteriophage,Frontiers in microbiology,6(2015)316.

[6]L.Specthrie,E.Bullitt,K.Horiuchi,P.Model,M.Russel,L.Makowski,Construction of amicrophage variant of filamentous bacteriophage,J Mol Biol,228(1992)720-724.

[7]U.Tsedev,C.W.Lin,G.T.Hess,J.N.Sarkaria,F.C.Lam,A.M.Belcher,Phage Particles of Controlled Length and Genome for In Vivo Targeted Glioblastoma Imaging and Therapeutic Delivery,ACS Nano,16(2022)11676-11691.

[8]K.Q.Han,G.Huang,C.-f.Gao,X.-l.Wang,B.Ma,L.-q.Sun,Z.-j.Wei,Identification of Lung Cancer Patients by Serum Protein Profiling Using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry,Am J Clin Oncol,31 133-139.

[9]M.Taheri,U.Saragovi,A.Fuks,J.Makkerh,J.Mort,C.P.Stanners,Self recognition in the Ig superfamily.Identification of precise subdomains in carcinoembryonic antigen required for intercellular adhesion,Journal of Biological Chemistry,275(2000)26935-26943.

[10]H.LJ,S.H,T.JA,O.S,N.H,S.JE,Expression of complementary DNA and genomic clones for carcinoembryonic antigen and nonspecific cross-reacting antigen in Chinese hamster ovary and mouse fibroblast cells and characterization of the membrane-expressed products,Cancer research,50(1990)2397-2403.

[11]I.Vogel,H.Francksen,E.Soeth,D.Henne-Bruns,H.Juhl,The carcinoembryonic antigen and its prognostic impact on immunocytologically detected intraperitoneal colorectal cancer cells,American Journal of Surgery,181(2001)188-193.

[12]H.HS,M.AM,Molecular Testing and the Pathologist's Role in Clinical Trials of Breast Cancer,Clinical breast cancer,16(2016)166-179.

disclosure of Invention

The invention aims at solving the existing problems and provides a CEA protein capable of recognizing the surface of a tumor, a short chain phagemid expressing a 3C protein, and construction and application thereof.

A CEA protein and 3C protein-expressing short chain phagemid capable of recognizing tumor surfaces, characterized in that: the short chain phagemid comprises:

the N-terminal insertion sequence of the pIII end of the auxiliary phage is CEA single-chain antibody (svFv), and a eukaryotic expression related element expression cassette containing SVV 3C gene is inserted into a phagemid vector pUC57-LS7 containing the replication origin and packaging sequence of M13 phage.

Can identify CEA protein on the surface of tumor and the application of short chain phagemid expressing 3C protein in targeting and oncolysis.

Construction of a short chain phagemid involves insertion of a single chain antibody sequence (scFv) of CEA at the N-terminus of the pIII gene of the recombinant phage, and insertion of a eukaryotic expression element ITR-cassette fragment between the replication origin and the packaging sequence of the phagemid vector pUC57-LS 7.

The target gene insertion site in the phagemid vector can be substituted for insertion of other target genes.

The sequence of the phage vector for constructing the recombinant phage vector and for pIII display is SEQ ID NO.1.

The CEA svFv is directly fused to the amino terminus of the pIII protein and the eukaryotic expression element ITR-cassette fragment is inserted between the phagemid vector pUC57-LS7 origin of replication and the packaging sequence.

A construction method of CEA protein capable of recognizing tumor surface and short chain phagemid expressing 3C protein is characterized in that: the method comprises the following steps:

(1) Taking a phage vector M13KO7 as a template, and respectively carrying out PCR amplification by adopting a primer 1, a primer 2, a primer 3 and a primer 4 to obtain a PCR product;

primer 1 sequence is SEQ ID NO.2:5'-ATTCCTTTAGTTGTTCCTTTCTATCAGGTTAAGCTGCAGCAGTC-3';

primer 2 has a sequence of SEQ ID NO.3:5'-AACAGTTTCAGCGGAGTGAGATTTCAGCTCGAGCTTGGTCCCAGC-3';

primer 3 sequence is SEQ ID NO.4:5'-TGTTCCTTTCTATTCTCACTCCCAGGTTAAGCTGCAGCAGTCTGG-3';

primer 4 has a sequence of SEQ ID NO.5:5'-AACAGTTTCAGCGGAGTGAGATTTCAGCTCGAGCTTGGTCCCAGC-3';

the primer 1 is a forward primer designed according to the upstream sequence of the CEA svFv; the primer 2 is a reverse primer designed according to the downstream sequence of the CEA svFv; the primer 3 is a forward primer designed according to the sequence upstream of the phage vector M13KO7 pIII; the primer 4 is a reverse primer designed according to the sequence downstream of the phage vector M13KO7 pIII;

(2) Carrying out recombination reaction on the two PCR products obtained in the step (1) through homologous recombination enzyme to obtain a recombinant phage vector M13KO7-ACEA svFv;

(3) Taking phagemid vectors pUC57-LS7 and pUC57-ITR-cassette as templates, and respectively carrying out PCR amplification by adopting a primer 5 and a primer 6 as well as a primer 7 and a primer 8 to obtain a PCR product;

primer 5 sequence is SEQ ID NO.6:5'-GCGCGCAGCTGCCTGCAGGCTTGGACGCGCCCTGTAGCGGC-3';

primer 6 has a sequence of SEQ ID NO.7:5'-CGCGCAGCTGCCTGCAGGCTTGCCCGAGATAGGGTTGAGTG-3';

primer 7 sequence is SEQ ID NO.8:5'-AACCCTATCTCGGGCAAGCCTGCAGGCAGCTGCGCGCTCG-3';

primer 8 has a sequence of SEQ ID NO.9:5'-TACAGGGCGCGTCCAAGCCTGCAGGCAGCTGCGCGCTCGC-3';

the primer 5 is a forward primer designed according to the sequence upstream of the phagemid vector pUC57-LS 7; the primer 6 is a reverse primer designed according to the sequence downstream of the phagemid vector pUC57-LS 7; the primer 7 is a forward primer designed according to the sequence upstream of the vector pUC 57-ITR-cassette; the primer 8 is a reverse primer designed according to the sequence downstream of the vector pUC 57-ITR-cassette;

(4) Carrying out recombination reaction on the two PCR products obtained in the step (3) through homologous recombination enzyme to obtain a phagemid vector pUC57-LS7-ITR-cassette;

(5) And (3) super-infecting pUC57-LS7-ITR-cassette with the recombinant phage vector M13KO7-ACEA svFv obtained in the step (2) to obtain a short-chain phagemid M13KO7-ACEA svFv-LS7-ITR.

The primer 1 is a single-stranded DNA molecule shown as SEQ ID NO. 2; the primer 2 is a single-stranded DNA molecule shown as SEQ ID NO. 3; the primer 3 is a single-stranded DNA molecule shown as SEQ ID NO. 4; the primer 4 is a single-stranded DNA molecule shown as SEQ ID NO. 5.

A method for targeting and oncolytic bifunctional short chain phagemid construction comprising the steps of:

(a) Inserting the coding genes of CEA svFv into the coding genes of p III protein respectively, and inserting ITR-cassette genes into eukaryotic expression elements ITR-cassette fragments into a phagemid vector pUC57-LS7 replication origin and a packaging sequence to obtain a short-chain phagemid M13KO7-ACEA svFv-LS7-ITR;

(b) Packaging the recombinant phage vector obtained in the step (a) to obtain recombinant phage; the recombinant phage is subjected to super infection of pUC57-LS7-ITR-cassette to obtain a short-chain phagemid M13KO7-ACEA svFv-LS7-ITR with the functions of targeting and expressing eukaryotic genes.

The short chain phagemid prepared by the method is provided.

By the present invention, it is an object of the present invention to provide a short chain phagemid M13KO7-ACEA svFv-LS7-ITR with the function of targeting and expressing eukaryotic genes. It is a further object of the present invention to provide the use of a short chain phagemid capable of expressing proteins in eukaryotic cells. It is a further object of the present invention to provide a method for constructing a short chain phagemid which is fast, accurate and easy to handle.

In the present invention, the gene encoding the p III protein is derived from phage vector M13KO7; the coding gene of the eukaryotic expression element is derived from a vector pUC57-ITR-cassette.

The sequence of the recombinant phage vector with the functions of targeting and expressing eukaryotic genes provided by the invention is sequence 1 in a sequence table.

(a) Inserting the coding gene of the CEA svFv into the coding gene of the p III protein respectively, inserting the ITR-cassette gene into the eukaryotic expression element ITR-cassette fragment between the replication origin and the packaging sequence of the phagemid vector pUC57-LS7 of any of claims 1-3, and obtaining a short-chain phagemid by superinfection;

In the invention, the CEA svFv sequence is shown as a sequence 10 in a sequence table; the ITR-cassette sequence is shown as a sequence 11 in a sequence table.

The short chain phagemid prepared by the method also belongs to the protection scope of the invention.

The invention integrates phage pIII display system and eukaryotic expression element for the first time, forms short chain phagemid with targeting and gene expression in eukaryotic cells, enriches phage morphology, and provides new tools and methods for biological and medical application.

Filamentous phage M13 was about 900nm long and about 8nm in diameter. The genome is a single-stranded circular DNA of about 7,000nt in length, enclosed in a cylindrical protein shell consisting of 2700 copies of the pVIII protein. Phage is used as a carrier to target solid tumors, but the efficiency of targeting tumor by head protein is low and nonspecific adsorption is serious because the tail of phage is too long. The project is to place a Sein-card valley virus (SVV) non-structural protein 3C in a eukaryotic expression frame, connect with an M13 phage packaging element, and simultaneously introduce a single-chain antibody sequence of carcinoembryonic antigen into a pIII gene of M13 phage to prepare a phagemid plasmid. After the phagemid plasmid is transformed into a host, co-transfecting helper phage M13KO7-ACEA scFv to obtain a short-chain phagemid which efficiently targets tumor cells, and identifying the short-chain phagemid by a transmission electron microscope, fluorescent observation and the like.

Drawings

FIG. 1 is a map of phage vector M13KO 7.

FIG. 2 is a map of phagemid vector pUC57-LS 7.

FIG. 3 is a map of recombinant phagemid vector pUC57-LS7-ITR-cassette

FIG. 4 is a schematic diagram of the construction of the short chain phagemid M13KO7-ACEA svFv-LS7-ITR

FIG. 5 is a transmission observation of the short chain phagemid M13KO7-ACEA svFv-LS7-ITR.

FIG. 6 shows the results of an indirect ELISA assay for short chain phagemid M13KO7-ACEA svFv-LS7-ITR.

Detailed Description

The invention is further described with reference to the accompanying drawings; the following examples are intended to illustrate, but not limit, the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Phage M13KO7: NEB company product with catalog number N0315S.

Coli ER2738: NEB corporation product, having catalog number E4104.

EXAMPLE 1 construction of phage vectors for targeting

In this example, the coding gene of CEA svFv is inserted into the coding gene of phage M13KO7 pIII protein, respectively, and ITR-cassette gene is inserted into eukaryotic expression element ITR-cassette fragment and inserted between the replication origin and packaging sequence of phagemid vector pUC57-LS7 according to any of claims 1-3, so as to obtain recombinant phage super-infected pUC57-LS7-ITR-cassette, and obtain short-chain phagemid M13KO7-ACEA svFv-LS7-ITR with targeting and eukaryotic gene expression functions.

The design idea is as follows: on phage vector M13KO7, the coding gene of CEA svFv was inserted after the amino-terminal signal peptide of pIII gene to obtain a recombinant phage vector with targeting.

1. Primer design

The primer 1 is a forward primer designed according to the upstream sequence of the CEA svFv; the primer 2 is a reverse primer designed according to the downstream sequence of the CEA svFv; the primer 3 is a forward primer designed according to the sequence upstream of the phage vector M13KO7 pIII; the primer 4 is a reverse primer designed according to the sequence downstream of the phage vector M13KO7 pIII. The sequence is as follows:

primer 1 (sequence 2): 5'-ATTCCTTTAGTTGTTCCTTTCTATCAGGTTAAGCTGCAGCAGTC-3';

primer 2 (sequence 3): 5'-AACAGTTTCAGCGGAGTGAGATTTCAGCTCGAGCTTGGTCCCAGC-3';

primer 3 (sequence 4): 5'-TGTTCCTTTCTATTCTCACTCCCAGGTTAAGCTGCAGCAGTCTGG-3';

primer 4 (sequence 5): 5'-AACAGTTTCAGCGGAGTGAGATTTCAGCTCGAGCTTGGTCCCAGC-3'.

2. Construction of recombinant phages for targeting

1. PCR amplification

PCR amplification is carried out by using CEA svFv and phage vector M13KO7 as templates and respectively using primer 1, primer 2, primer 3 and primer 4 to obtain PCR products containing coding genes of recombinant pIII proteins for targeting and CEA svFv.

PCR reaction system (50 μl): phanta Max Super-Fidelity DNA Polymerase enzyme 1 μl; template (1 ng/. Mu.l) 1. Mu.l; dNTP Mix 1. Mu.l; 2X Phanta Max Buffer. Mu.l; (1. Mu.l each of primer 1 and primer 2 or primer 3 and primer 4 (10. Mu.M; 20. Mu.l of sterile water).

PCR reaction procedure: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 15s, annealing at 62℃for 15s, elongation at 72℃for 10min/1min,35 cycles, respectively; extending for 5min at 72 ℃.

After the reaction, the PCR product was detected by agarose gel electrophoresis.

2. Homologous recombination

The target fragment obtained by PCR amplification is subjected to a specification homologous recombination reaction.

Homologous recombination reaction System (20. Mu.l): 4 μl of phage M13KO7 fragment; 10 μl of CEA svFv fragment; 5 XCE II Buffer 4. Mu.l; exnaseII 2. Mu.l. The reaction was carried out at 37℃for 30min. After the reaction was completed, the reaction tube was immediately cooled in an ice-water bath for 5min.

3. Transformation

Transforming the recombinant reaction product obtained in the step 2 into competent cells of escherichia coli Trans1-Blue Chemically Competent Cell, and coating a Kan plate for overnight culture at 37 DEG C

4. Picking up monoclonal identification

Monoclonal M13KO7-ACEA svFv was selected, cultured and PCR was performed using primers 1 and 2 (SEQ ID NO: 1). The PCR was initially used to identify the correct vector for sample sequencing.

The phage with correct sequence is the recombinant phage M13KO7-ACEA svFv for targeting.

Example 2, example 1 application examples of the resulting short chain phagemid for targeting and expression

The ITR-cassette gene fragment is inserted between a phagemid vector pUC57-LS7 replication origin and a packaging sequence to obtain a recombinant phagemid vector pUC57-LS7-ITR-cassette, and a short-chain phagemid M13KO7-ACEA svFv-LS7-ITR with the functions of targeting and expressing eukaryotic genes is obtained through recombinant phage superinfection. The method comprises the following steps:

the design idea is as follows: the ITR-cassette gene fragment is inserted into a phagemid vector pUC57-LS7 through homologous recombination, and a short-chain phagemid with the functions of targeting and expressing eukaryotic genes is obtained through recombinant phage superinfection.

1. Primer design

The primer 5 is a forward primer designed according to the sequence upstream of the phagemid vector pUC57-LS 7; the primer 6 is a reverse primer designed according to the sequence downstream of the phagemid vector pUC57-LS 7; the primer 7 is a forward primer designed according to the sequence upstream of the vector pUC 57-ITR-cassette; the primer 8 is a reverse primer designed based on the sequence downstream of the vector pUC57-ITR-cassette. The sequence is as follows:

primer 5 (sequence 6): 5'-GCGCGCAGCTGCCTGCAGGCTTGGACGCGCCCTGTAGCGGC-3';

primer 6 (sequence 7): 5'-CGCGCAGCTGCCTGCAGGCTTGCCCGAGATAGGGTTGAGTG-3';

primer 7 (sequence 8): 5'-AACCCTATCTCGGGCAAGCCTGCAGGCAGCTGCGCGCTCG-3';

primer 8 (sequence 9): 5'-TACAGGGCGCGTCCAAGCCTGCAGGCAGCTGCGCGCTCGC-3'.

2. Construction of recombinant phagemid vectors

1. PCR amplification

And taking the phagemid vector pUC57-LS7 and the vector pUC57-ITR-cassette as templates, and respectively adopting a primer 5, a primer 6, a primer 7 and a primer 8 for PCR amplification to obtain PCR products of the phagemid vector pUC57-LS7 fragment and the ITR-cassette fragment.

2. Homologous recombination

Homologous recombination reaction System (20. Mu.l): 4 μl of the phagemid vector pUC57-LS7 fragment; 10 μl of ITR-cassette fragment; 5 XCE II Buffer 4. Mu.l; exnaseII 2. Mu.l. The reaction was carried out at 37℃for 30min. After the reaction was completed, the reaction tube was immediately cooled in an ice-water bath for 5min.

3. Transformation

And (3) transforming the recombinant reaction product obtained in the step (2) into escherichia coli DH5 alpha competent cells, and then coating an Amp plate for overnight culture at 37 ℃.

4. Picking up monoclonal identification

The monoclonal pUC57-LS7-ITR-cassette was picked, cultured and subjected to PCR verification using primers 7 and 8 (sequences as above). The PCR was initially used to identify the correct vector for sample sequencing.

3. Short chain phagemid packaging, characterization and targeted identification

1. Recombinant phage packaging

The successfully sequenced positive recombinant phage vector M13KO7-ACEA svFv clone was inoculated into 5ml of LB liquid medium containing tetracycline resistance, and cultured overnight. The next day, after phage amplification culture was performed for 24 hours by transferring the culture broth into 500ml LB liquid medium, centrifugation was performed at 10000rpm at 4℃for 30 minutes, and the supernatant was collected. 1/6 volume of 16.7% PEG/NaCl (formulation: solvent water, solute and concentration of 16.7g/100ml PEG8000,3.3M NaCl) was added and left standing overnight at 4 ℃. Then, the mixture was centrifuged at 12000rpm at 4℃for 20 minutes, and the precipitate was collected. Continuing, after suspending with 1ml of sterile water, centrifugation was carried out at 12000rpm for 5min at 4℃to remove impurities, and the supernatant was transferred to a new EP tube. Again, 1/6 volume of 16.7% PEG/NaCl (formulation above) was added and left standing at room temperature for 1h. Finally, centrifuging at 12000rpm and 4 ℃ for 20min, and adding sterile water or PBS buffer solution with proper volume for suspension to obtain the recombinant phage. The concentration of phage can be determined by spectrophotometry.

2. Short chain phagemid packaging

E.coli cultures containing pUC57-LS7-ITR-cassette were infected with recombinant helper phage M13-ACEA scFv, and the supernatant obtained after purification with 15% and 4% PEG was concentrated by dialysis to obtain the short chain phagemid M13KO7-ACEA svFv-LS7-ITR.

3. Characterization of recombinant phages

The purified short-chain phagemid M13KO7-ACEA svFv-LS7-ITR was diluted 100-fold, 50. Mu.L was pipetted and fixed onto a 200 mesh copper mesh for 10min, and after 90s of 1% phosphotungstic acid, dried for 20min. The samples were observed by Tecnai 12 transmission electron microscopy.

4. pIII display detection

(1) pIII detection

1) Antigen coating liquid (Na) ₂ CO ₃ 0.159g,NaHCO ₃ 0.293g, fixed to 100mL, pH=9.6) carcinoembryonic antigen protein was diluted to 2. Mu.g/mL, the dilutions were added to 96-well plates at 100. Mu.L per well and coated overnight at 4 ℃.

2) The next day, the extra carcinoembryonic antigen protein dilutions in 96-well plates were discarded, and 1XPBS (1 xBS, v/v with 0.05% Tween 80) was washed 3 times and dried.

3) 200. Mu.L of 1% BSA (v/v) was added, and after blocking at 37℃for 2 hours, 1xPBST was washed three times and dried.

4) 100. Mu.L of 100-fold dilutions of M13KO7-ACEA scFv and short-chain phagemid M13KO7-ACEA svFv-LS7-ITR were added, respectively, and incubated at 37℃for 1h.

5) After washing for 6 times with 1xPBST, it was dried, 100. Mu.L of anti-M13 phage capsid protein g8p antibody 100-fold diluted with 1xPBS was added and incubated at 37℃for 1h.

6) After washing 1xPBST 6 times, it was dried, 100. Mu.L of goat anti-mouse IgG HRP 10000-fold diluted with 1xPBS was added, and incubated at 37℃for 30min.

7) Washing for 6 times by using 1xPBST, drying, adding 100 mu L of TMB color reagent, and reacting for 5-10min.

8) 50. Mu.L of stop solution (2 m H2SO 4) was added thereto, and the absorbance at OD450nm was measured.

The above results demonstrate that the phage vector for targeting constructed in example 2 can successfully display ACEA scFv simultaneously.

(2) EGFP detection

HT29 cells were seeded in 12-well plates, cultured overnight, and phage M13KO7-ACEA scFv and short-chain phagemid M13KO7-ACEA svFv-LS7-ITR (final concentration 10) ⁶ PFU/cell) was observed under a fluorescence microscope 24h after infection.

Claims

1. A CEA protein and 3C protein-expressing short chain phagemid capable of recognizing tumor surfaces, characterized in that: the short chain phagemid comprises:

2. The use of CEA protein recognizing tumor surface and short chain phagemid expressing 3C protein according to claim 1 for targeting and oncolysis.

3. The use according to claim 2, wherein: construction of a short chain phagemid involves insertion of a single chain antibody sequence (scFv) of CEA at the N-terminus of the pIII gene of the recombinant phage, and insertion of a eukaryotic expression element ITR-cassette fragment between the replication origin and the packaging sequence of the phagemid vector pUC57-LS 7.

4. A use according to claim 3, wherein: the target gene insertion site in the phagemid vector can be substituted for insertion of other target genes.

5. Use according to any one of claims 2-4, characterized in that: the sequence of the phage vector for constructing the recombinant phage vector and for pIII display is SEQ ID NO.1.

6. The use according to claim 2, characterized in that: the CEA svFv is directly fused to the amino terminus of the pIII protein and the eukaryotic expression element ITR-cassette fragment is inserted between the phagemid vector pUC57-LS7 origin of replication and the packaging sequence.

7. A construction method of CEA protein capable of recognizing tumor surface and short chain phagemid expressing 3C protein is characterized in that: the method comprises the following steps:

8. The method according to claim 7, wherein: the primer 1 is a single-stranded DNA molecule shown as SEQ ID NO. 2; the primer 2 is a single-stranded DNA molecule shown as SEQ ID NO. 3; the primer 3 is a single-stranded DNA molecule shown as SEQ ID NO. 4; the primer 4 is a single-stranded DNA molecule shown as SEQ ID NO. 5.

9. A method for targeting and oncolytic bifunctional short chain phagemid construction, characterized by: the method comprises the following steps:

10. A short chain phagemid prepared by the method of claim 9.