CN117660372A - Application of recombinant Newcastle disease virus encoding long-acting chicken infectious bursal protective antigen in the preparation of two-part vaccine - Google Patents

Abstract

The invention discloses an application of newcastle disease recombinant virus for encoding long-acting chicken infectious bursal disease protective antigen in preparing bivalent vaccine. The invention provides a Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF; VP2DS-Fc in Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF is as follows (a) or (b): (a) A protein consisting of amino acid residues 1-531 of sequence 1; (b) A protein having the same activity obtained by substituting and/or deleting and/or adding the amino acid residue in the step (a); the chGM-CSF in the recombinant Newcastle disease virus rClone30-VP2DS-Fc-chGM-CSF is either (c) or (d) as follows: (c) Protein consisting of 1 st to 144 th amino acid residues from the N terminal of a sequence 3 in a sequence table; (d) A protein derived from the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the step (c) and has the same activity; the recombinant virus rClone30-VP2DS-Fc-chGM-CSF of the newcastle disease at different sites can strengthen humoral immunity and inflammatory response of organisms for a long time, and opens up a new situation in the prevention history of newcastle disease and infectious bursal disease of chickens.

Description

Newcastle disease recombinant virus encoding long-acting chicken infectious bursal protective antigen is in production Application in preparation of two-part vaccine

Technical field

The present invention relates to the application of Newcastle disease recombinant virus encoding long-acting chicken infectious bursal of Fabricius protective antigen in the preparation of a two-combination vaccine.

Background technique

Infectious bursal disease (IBD) is an acute, highly contagious viral infection caused by infectious bursal disease virus (IBDV). It can attack B lymphocytes in the bursa of Fabricius, causing chicken Immunosuppression increases the susceptibility of chickens to other microorganisms and increases the risk of vaccination failure. Currently, IBD has been listed as a major disease in the poultry industry by the International Organization for Animal Health (OIE). IBDV is highly stable in the environment and difficult to inactivate. It is difficult to completely remove IBDV from farms with IBD outbreaks. Therefore, vaccination is still the first choice prevention measure to effectively control the epidemic of IBD. Currently, the vaccines commonly used on the market to prevent IBD are mainly attenuated vaccines, moderately virulent vaccines, inactivated vaccines and subunit vaccines. The protective ability of attenuated vaccines is weak, and there is still the possibility of being infected with IBDV after immunization; the moderately virulent vaccines will cause serious damage to the bursa of Fabricius; the antigen content of inactivated vaccines must reach a high level to induce chickens to produce strong IBDV Humoral immunity; the immune effect of subunit vaccines is greatly affected by the vaccine preparation process, and the serum half-life in the body is relatively short. Therefore, there is an urgent need to develop a new vaccine that is more efficient in producing antibodies.

Fc fusion protein is composed of the Fc region of an IgG antibody and a foreign protein. Its Fc fragment can bind to the neonatal Fc receptor (FcRn), prolonging the serum half-life of the foreign protein in the body, thereby improving the response of immune effector cells to the antigen. capture efficiency. The first therapeutic Fc fusion protein was used to treat AIDS. Currently, 11 Fc fragment fusion proteins have been approved by the FDA, and many new Fc fusion proteins are in preclinical and clinical development. stage. The avian Ig Y Fc region and the mammalian Ig G Fc fragment not only have similar structural compositions, but also have similar binding properties between them. Therefore, using Ig Y Fc to improve the immunogenicity of target proteins is theoretically a feasible way.

Newcastle disease (ND) is an acute highly contagious infectious disease that mainly infects poultry, causing the host to have difficulty breathing, persistent high fever, tissue and gastrointestinal mucosal bleeding, or central nervous system disorder. Although ND is prevented through vaccination in my country, due to virus strain mutation or vaccine immunity failure, the disease is still an important factor restricting poultry breeding, and it is still one of the major poultry diseases that harms the poultry industry. The cause of ND is the NDV virus, an envelope-coated, non-segmented negative-strand RNA virus with a genome of 15.2kb in length, encoding six structural proteins in sequence, including nucleocapsid protein (NP) and phosphoprotein. (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L). Each gene of a specific structural protein has an initiation factor and a termination factor sequence. Each protein gene is separated from each other by intergenic sequences to ensure the accuracy of its expression. Based on the characteristics of the NDV genome, inserting one or more foreign genes into its genome does not affect the biological characteristics of NDV and has good safety and effectiveness. Therefore, it has great advantages in being used as a vaccine carrier to develop bivalent vaccines against important poultry pathogens.

By introducing long-acting chicken infectious bursal protective antigen (VP2DS-Fc) and granulocyte-macrophage colony-stimulating factor (chCM-CSF) genes into different sites of the Newcastle disease virus genome, the present invention can improve the body's health in the long term. The immune response to the vaccine provides a reference for the development of vaccines against other infectious viral diseases. At the same time, the selected insertion site does not affect the proliferation ability of NDV, that is, it will not affect subsequent industrial production performance.

Contents of the invention

One of the purposes of the present invention: to provide Newcastle disease recombinant virus rClone30-VP2DS-Fc encoding long-acting chicken infectious bursal protective antigen (VP2DS-Fc) and granulocyte-macrophage colony-stimulating factor (chCM-CSF) -IRES-GM-CSF (P/M) (the VP2DS-Fc gene and GM-CSF are inserted between the P and M genes of the attenuated Newcastle disease strain Lasota in the form of VP2DS-Fc-IRES-GM-CSF) and rClone30 -VP2DS-Fc(NP)-GM-CSF(P/M) (The VP2DS-Fc gene is inserted into the NP gene of the attenuated Newcastle disease strain Lasota and the biological adjuvant GM-CSF is inserted into the P gene of the attenuated Newcastle disease strain Lasota. and M gene).

The second object of the present invention is to provide the application of recombinant virus rClone30-VP2DS-Fc-GM-CSF at different sites in preventing chicken Newcastle disease and chicken infectious bursal disease. After the chickens are immunized with the vaccine, the chickens can quickly produce antibodies, and the half-life of the antibodies is extended, which makes up for the shortcomings of the existing Newcastle disease and infectious bursal disease vaccines that take too long to produce antibodies, and can also enhance the immune effect of the vaccine.

The Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF at different sites and its genome nucleotide sequence, protein sequence and VP2DS-Fc gene are inserted into the Newcastle disease recombinant virus rClone30-GM-chCSF, and the method thereof Products used to prevent Newcastle disease and/or chicken infectious bursal disease are within the scope of protection of the present invention.

The Newcastle disease recombinant virus rClone30-VP2DS-Fc-IRES-GM-CSF (P/M) constructed by the present invention has a living vector whose basic skeleton is the Lasota attenuated strain, and VP2DS-Fc-IRES is inserted between its P gene and M gene. -GM-CSF gene; the constructed Newcastle disease recombinant virus rClone30-VP2DS-Fc(NP)-GM-CSF(P/M), the basic skeleton of the living vector is the Lasota attenuated strain, and the VP2DS-Fc gene and the VP2DS-Fc gene are inserted into the NP gene The GM-CSF gene is inserted between its P gene and M gene. The VP2DS-Fc of the Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF is as follows (a) or (b): (a) consists of amino acid residues 1-531 from the N terminus of Sequence 1 in the sequence list The protein; (b) the recombinant virus derived from (a) that has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same activity is within the protection scope of the present invention; the GM-CSF in Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF is as follows (c) or (d): (c) A protein composed of amino acid residues 1-144 from the N terminus of Sequence 3 in the sequence list ; (d) Recombinant viruses derived from (c) that have undergone the substitution and/or deletion and/or addition of one or several amino acid residues and have the same activity are within the scope of the present invention;

The VP2DS-Fc and GM-CSF genes encoding the Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF also belong to the protection scope of the present invention.

In the gene, the DNA molecule encoding the VP2DS-Fc is as follows (1) or (2) or (3): (1) Sequence 2 of the sequence list is shown as nucleotides 1-1593 from the 5' end. DNA molecules; (2) DNA molecules that hybridize to the DNA sequence defined in (1) under stringent conditions and encode proteins with the same activity; (3) have at least 90% homology with the DNA sequence defined in (1) And DNA molecules encoding proteins with the same activity.

The DNA molecule encoding the GM-CSF is as follows (4) or (5) or (6): (4) The DNA molecule represented by nucleotides 1-432 from the 5' end of Sequence 4 in the sequence list; ( 5) DNA molecules that hybridize to the DNA sequence defined in (4) under stringent conditions and encode proteins with the same activity; (6) have at least 90% homology with the DNA sequences defined in (4) and encode proteins with the same activity of protein DNA molecules.

The above stringent conditions can be hybridization in a solution of 6×SSC, 0.5% SDS at 65°C, and then washing the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Expression cassettes, recombinant vectors, transgenic cell lines or recombinant viruses containing any of the above genes all fall within the protection scope of the present invention.

The related preparations of Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF at different sites are within the scope of protection of the present invention.

The recombinant Newcastle disease virus rClone30-VP2DS-Fc-GM-CSF constructed in the present invention at different sites can be regarded as a genetically engineered attenuated strain, and its corresponding preparations, such as freeze-dried powder injection preparations, liquid biological preparations, etc. It falls within the scope of this patent protection.

The Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF at different sites and its genome nucleotide sequence and protein sequence are within the protection scope of the present invention.

The application of the Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF at different sites in preventing Newcastle disease and infectious bursal disease falls within the protection scope of the present invention.

The application of the Newcastle disease recombinant virus rClone30-chGM-CSF in preventing other viral infectious diseases in chickens falls within the protection scope of the present invention.

The invention provides Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF encoding fusion protein VP2DS-Fc, and the anti-NDV antibody level, anti-IBDV antibody level and IL-4 produced by the rClone30-VP2DS-Fc-chGM-CSF group The contents were higher than those in the rClone30 control group. It shows that the rClone30-VP2DS-Fc-chGM-CSF vaccine can enhance the body's humoral immunity level and inflammatory response.

Description of drawings

Figure 1 shows rClone30-VP2DS-Fc-GM-CSF viral hemagglutination (HA) at different sites.

Figure 2 shows the use of ELISA kits to detect the expression of foreign proteins in the cell supernatant of DF-1 cells infected with rClone30-VP2DS-Fc-GM-CSF at different sites. Picture A shows the VP2DS-Fc protein expression level, and Picture B shows the GM-CSF protein expression level.

Figure 3 shows the dynamic growth curve of rClone30-VP2DS-Fc-chGM-CSF at different sites in infected cells.

Figure 4 shows experimental SPF chickens immunized with rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M), rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M), rClone30-VP2DS-Fc, rClone30 -NDV antibody titers at different time points after GM-CSF and rClone30.

Figure 5 shows experimental SPF chickens immunized with rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M), rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M), rClone30-VP2DS-Fc, rClone30 -IBDV antibody titers at different time points after GM-CSF and rClone30.

Figure 6 shows experimental SPF chickens immunized with rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M), rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M), rClone30-VP2DS-Fc, rClone30 -IL-4 content in serum at different time points after GM-CSF and rClone30.

Specific implementation plan:

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

Example 1: Construction of pBrClone30-VP2DS-Fc-GM-CSF at different sites

1.1Construction of pBrClone30-VP2DS-Fc-IRES-GM-CSF(P/M)

Overlap PCR was used to connect VP2DS-Fc, IRES and chGM-CSF genes together, and a gel recovery kit was used to recover the fragments and connected to the T vector to construct the recombinant plasmid VP2DS-Fc-IRES-chGM-CSF-T. VP2DS-Fc-IRES-chGM-CSF-T and pBrClone30 were double digested using Sac II and PmeI. The fragments and vectors were recovered separately using a gel recovery kit, and the VP2DS-Fc-IRES-chGM-CSF fragment was connected to the rClone30 vector to construct the recombinant plasmid pBrClone30-VP2DS-Fc-IRES-chGM-CSF (P/M).

1.2 Construction of pBrClone30-VP2DS-Fc(NP)-GM-CSF(P/M)

Sac II and Pme I were used to double digest pUC18-chGM-CSF and pBrClone30. Use a gel recovery kit to recover the fragments and vector respectively, connect the chGM-CSF fragment to the rClone30 vector, and construct the recombinant plasmid pBrClone30-chGM-CSF.

PCR IRES-VP2DS-Fc gene and use Aat II and Apa I to digest pBrClone30-chGM-CSF. A gel recovery kit was used to recover the fragments and vectors respectively, and the IRES-VP2DS-Fc fragment was connected to the pBrClone30-chGM-CSF vector using a homologous recombination kit to construct the recombinant plasmid pBrClone30-VP2DS-Fc(NP)-chGM-CSF( P/M).

Example 2: Rescue and HA detection of rClone30-VP2DS-Fc-GM-CSF at different sites

2.1 Rescue of rClone30-VP2DS-Fc-IRES-GM-CSF (P/M): Combine recombinant NDV rClone30-VP2DS-Fc-IRES-GM-CSF (P/M) with three helper plasmids pTM-NP, pTM- P and pTM-L were co-transfected into BHK-21 cells. 72 hours after transfection, the supernatant of the transfected cells was harvested, frozen and thawed three times, and then inoculated into the allantoic cavity of 9-11 day-old SPF chicken embryos. After 72 hours of constant temperature culture, the chicken embryo allantoic fluid was harvested, centrifuged and then subjected to chicken red blood cell agglutination (HA). The allantoic fluid with positive results was continuously passaged three times with SPF grade chicken embryos and then mixed.

After HA testing, the results showed that Figure 1: HA titer was 512. The recombinant virus rClone30-VP2DS-Fc-IRES-GM-CSF(P/M) will be successfully rescued.

2.2 Rescue of rClone30-VP2DS-Fc(NP)-GM-CFS(P/M): Combine recombinant NDV rClone30-VP2DS-Fc(NP)-GM-CFS(P/M) with three helper plasmids: pTM-NP, pTM-P and pTM-L were co-transfected into BHK-21 cells. 72 hours after transfection, the supernatant of the transfected cells was harvested, frozen and thawed three times, and then inoculated into the allantoic cavity of 9-11 day-old SPF chicken embryos. After 72 hours of constant temperature culture, the chicken embryo allantoic fluid was harvested, centrifuged and then subjected to chicken red blood cell agglutination (HA). The allantoic fluid with positive results was continuously passaged three times with SPF grade chicken embryos and then mixed.

After HA testing, the results showed that Figure 1: HA titer was 512. The recombinant virus rClone30-VP2DS-Fc(NP)-GM-CFS(P/M) will be successfully rescued.

Example 3: ELISA detection of expression levels of exogenous genes VP2DS-Fc and chGM-CSF

DF-1 cells in the logarithmic growth phase were inoculated into six-well plates, and chicken embryos were inoculated with allantoic fluid virus diluted in appropriate multiples in DMEM and 0.1 MOI of recombinant rClone30-VP2DS-Fc-IRES-chGM-CSF (P/M). , rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M) and rClone30 were used to infect the cells respectively at 37°C. After incubating for 1 hour, the cells were washed three times with DMEM, and then complete DMEM was added to continue culturing. After 48 hours, the cells were frozen and thawed three times, and the cell supernatant was taken to detect the expression of exogenous genes VP2DS-Fc and chGM-CSF. The specific steps should be followed according to the instructions of the kit. After adding 50 μL of stop solution (2 mol/L H ₂ SO4), use a microplate reader to detect the OD value at a wavelength of 450 nm. Set rClone30 as the control group.

The results are shown in Figure 2. The ELISA results show that rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M) and rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M) can stably express VP2DS-Fc. and chGM-CSF foreign protein.

Example 4: Stable proliferation analysis of rClone30-VP2DS-Fc-chGM-CSF cells at different sites

When the DF-1 cells are cultured to the logarithmic phase, they are inserted into a six-well cell plate, and the viruses are inoculated onto the cell monolayer at a dose of 0.1 MOI. Complete the cells with DMEM containing 5% CS and 1 μg/mL trypsin. Culture medium serves as cell culture fluid. Place in 5% CO2 and continue to culture at 37°C for several days. The cell supernatant is harvested every 24 hours, and its TCID ₅₀ is measured respectively. As shown in Figure 3, the recombinant NDV rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M), rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M) carrying foreign genes and NDV rClone30 The parental strains maintain consistent reproductive titers.

Example 5: Determination of NDV antibody titers after immunizing chickens with rClone30-VP2DS-Fc-chGM-CSF at different sites

14-day-old SPF chickens were randomly divided into 6 groups, with 10 birds in each group. rClone30, rClone30-GM-CSF, rClone30-VP2DS-Fc, rClone30-VP2DS-Fc-IRES-chGM-CSF(P/M) and rClone30-VP2DS-Fc(NP)-chGM-CSF(P/M) respectively. 10 ⁵ TCID ₅₀ /0.1ml was immunized by intramuscular injection at a dose of 200 μL/animal. The blank group was replaced with the same dose of 0.9% normal saline. Blood was collected from the wing veins of chickens in each group on days 7, 14, 21, and 28 days after immunization to separate serum, and the NDV antibody titers were detected according to conventional methods. The results are shown in Figure 4.

Example 6: Determination of IBDV antibody titer after immunizing chickens with rClone30-VP2DS-Fc-chGM-CSF at different sites

Blood was collected from the wing veins of chickens in each group on days 5, 7, 14, 21, and 28 days after immunization to separate serum, and the IBDV antibody titer was detected according to the ELISA method. The results are shown in Figure 5.

Example 7: Determination of serum IL-4 content after immunizing chickens with rClone30-VP2DS-Fc-chGM-CSF at different sites

Blood was collected from the wing veins of chickens in each group on days 7, 10, 14, and 21 days after immunization to separate serum, and the IL-4 content in the serum was detected according to the instructions of the ELISA kit. The results are shown in Figure 6.

Claims (9)

1. Newcastle disease recombinant virus rClone30-VP2DS-Fc-GM-CSF. The recombinant virus rClone30-VP2DS-Fc-IRES-GM-CSF (P/M) has a live vector basic skeleton of Lasota classical vaccine strain, and VP2DS-Fc-IRES-GM-CSF gene is inserted between P gene and M gene; the recombinant virus rClone30-VP2DS-Fc (NP) -chGM-CSF (P/M) has Lasota classical vaccine strain as the basic skeleton of its active carrier, and chGM-CSF gene inserted between its NP gene and VP2DS-Fc gene and its P gene and M gene.

VP2DS-Fc in the novel Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF is (a) or (b) as follows: (a) Protein consisting of amino acid residues 1-531 from the N terminal of a sequence 1 in a sequence table; (b) A protein derived from the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues and has the same activity; the chGM-CSF in the novel newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF is (c) or (d) as follows: (c) Protein consisting of 1 st to 144 th amino acid residues from the N terminal of a sequence 3 in a sequence table; (d) And (c) a protein derived from the same and having the same activity, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues.

2. A gene encoding the recombinant newcastle disease virus rcone 30-VP2DS-Fc-chGM-CSF of claim 1.

3. The gene of claim 2, wherein: in the gene, the DNA molecule encoding the Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF VP2DS-Fc is (1) or (2) or (3): (1) DNA molecules shown in 1 st to 1593 nd nucleotides from the 5' end of the sequence 2 in the sequence table; (2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (1) and which encodes a protein having the same activity; (3) A DNA molecule having at least 90% homology with the DNA sequence defined in (1) and encoding a protein having the same activity;

in the gene, the DNA molecule encoding the Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF chGM-CSF is (4) or (5) or (6) as follows: (4) DNA molecules shown in 1 st to 432 th nucleotides from the 5' end of a sequence 4 in a sequence table; (5) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (4) and which encodes a protein having the same activity; (6) A DNA molecule having at least 90% homology with the DNA sequence defined in (4) and encoding a protein having the same activity.

4. Expression cassettes, recombinant vectors, transgenic cell lines or recombinant viruses containing any of the above genes are within the scope of the present invention.

5. Use of the recombinant newcastle disease virus rcone 30-VP2DS-Fc-chGM-CSF according to claim 1 and the recombinant newcastle disease virus rcone 30-VP2DS-Fc-chGM-CSF gene according to claim 2 for the preparation of related products.

6. Use of the recombinant newcastle disease virus rcone 30-VP2DS-Fc-chGM-CSF of claim 1 and the recombinant newcastle disease virus rcone 30-VP2DS-Fc-chGM-CSF gene of claim 2 for the prevention of newcastle disease and/or infectious bursal disease in chickens.

7. Use of the newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF of claim 1 and the newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF gene of claim 2 in the preparation of novel biological adjuvant bivalent vaccines.

8. The genome nucleotide sequence and the protein sequence of the Newcastle disease recombinant virus rClone30-VP2DS-Fc-chGM-CSF at different sites are the protection scope of the invention.

9. The application of the newcastle disease recombinant virus and the construction mode thereof in the aspect of preventing infectious diseases of other viruses of chickens is the protection scope of the invention.