Method for reducing PRV virulence through gene insertion and application of method in vaccine preparation

Abstract

The invention provides a method for reducing PRV virulence through gene insertion and application of the method in vaccine preparation, and belongs to the technical field of gene engineering. The invention provides a method for reducing the virulence of a pseudorabies virus. An EGFP (Enhanced Green Fluorescent Protein) protein expression cassette is inserted into the upstream of a UL40 gene. According to the invention, after the PRV is subjected to the genetic engineering modification, the pseudorabies virus attenuated strain EGFP-PRV can be obtained through virus rescue. Compared with a wild type PRV-DX, the pathogenicity is weakened, no pathological clinical symptom appears after a Balb / c mouse is infected with the pseudorabies virus, and the pseudorabies vaccine has the prospect of preparing the pseudorabies vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a method for reducing the virulence of PRV by gene insertion and its application in the preparation of vaccines. Background Art

[0002] Porcine pseudorabies (PR) is an acute infectious disease of pigs caused by porcine pseudorabies virus (PRV), and is one of the major infectious diseases endangering the global pig industry, causing huge economic losses to the world pig industry. Since there is no effective treatment drug, the most effective prevention and control measure for this disease is still mainly the immunization of vaccines. Through the use of the vaccine strain Bartha K61 and the combination of relevant prevention and control measures, many European and American countries have completed the purification of this disease. However, since 2011, the emergence and prevalence of the genetically type II variant strains have severely damaged the protective effect of the Bartha K61 vaccine strain. This variant strain has a faster onset speed, more severe clinical symptoms, and a higher mortality rate after infecting pigs, and this strain can infect humans and cause severe clinical symptoms, threatening public health safety. Therefore, it is necessary to develop new vaccine strains for the wild genetically type II pseudorabies virus, and then do a good job in the prevention and control of genetically type II PRV.

[0003] Attenuated vaccine strains retain most of the natural antigenic epitopes of the pathogen and have good effects in the body's immune response, especially the T cell-mediated immune response. At present, there are research reports on attenuated strains of genetically type II PRV, which are basically obtained by using genetic engineering technology to delete the gene fragments related to virulence in the PRV genome. These candidate attenuated vaccine strains have clear deletion sites, can be used for differential diagnosis with the infected wild strains, and are not prone to virulence reversion. They are also known as serological "marker" vaccines and have played an important role in the prevention, control, and eradication of pseudorabies.

[0004] Statistics show that the virus genes currently undergoing deletion transformation mainly include gE (encoded by the virus gene US8), gI (encoded by the virus gene US7), TK (encoded by the virus gene UL23), gD (encoded by the virus gene US6), gG (encoded by the virus gene US4), etc., including the rescue of viruses with single-gene, double-gene, triple-gene, and quadruple-gene deletions. In addition, virus genes such as US2, US3, and US9 can also be deleted for the preparation of attenuated live vaccines. It is worth noting that the preparation strategies of these PRV attenuated live vaccines are all to delete the sequences of one or more functional genes in the virus genome. However, little is known about whether a PRV attenuated strain can be obtained by inserting a gene sequence without affecting the integrity of the virus genome. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for reducing the virulence of PRV by gene insertion and its application in the preparation of vaccines, and the purpose of reducing pathogenicity can be achieved by inserting an EGFP protein expression cassette upstream of the UL40 gene.

[0006] The first object of the present invention is to provide a method for reducing the virulence of pseudorabies virus, including inserting an EGFP protein expression cassette upstream of the UL40 gene of pseudorabies virus.

[0007] In a preferred embodiment of the present invention, it includes using the UL39 gene as the homologous left arm, and 9 bases upstream of UL40 and the UL40 gene as the homologous right arm, and inserting the EGFP protein expression cassette between the homologous left arm and the homologous right arm.

[0008] In a preferred embodiment of the present invention, the EGFP protein expression cassette includes CMV, EGFP, and SV40 connected in sequence.

[0009] In a preferred embodiment of the present invention, the EGFP protein expression cassette includes the nucleotide sequence shown in SEQ ID No.1.

[0010] The second object of the present invention is to provide an attenuated or avirulent pseudorabies virus mutant strain constructed by using the above method.

[0011] In a preferred embodiment of the present invention, it includes using the porcine pseudorabies virus PRV-DX as the parental strain, and constructing the attenuated or avirulent pseudorabies virus mutant strain after mutation;

[0012] The porcine pseudorabies virus PRV-DX has been deposited, and the deposit number is CGMCC No.14326.

[0013] The third object of the present invention is to provide a production method of the above attenuated or avirulent pseudorabies virus mutant strain, including inserting an EGFP protein expression cassette upstream of the UL40 gene of pseudorabies virus, and obtaining the attenuated or avirulent pseudorabies virus mutant strain through virus rescue.

[0014] In a preferred embodiment of the present invention, when performing virus rescue, vero cells are used as the host cells.

[0015] The fourth object of the present invention is to provide the application of the above attenuated or avirulent pseudorabies virus mutant strain in the preparation of pseudorabies vaccines.

[0016] The fifth object of the present invention is to provide a pseudorabies vaccine, including the above attenuated or avirulent pseudorabies virus mutant strain as an active ingredient, and also including excipients.

[0017] Beneficial effects: The present invention provides a method for genetically engineering a PRV strain. By inserting an EGFP protein expression cassette upstream of the UL40 gene, a pseudorabies virus attenuated strain EGFP-PRV with reduced pathogenicity can be constructed. After the above genetic engineering of PRV, the pseudorabies virus attenuated strain EGFP-PRV can be obtained through virus rescue. In the examples of the present invention, the type II pseudorabies virus strain PRV-DX (CGMCC No. 14326) is used as the parental strain, the UL39 gene is used as the homologous left arm, and 9 bases upstream of UL40 and the UL40 gene are used as the homologous right arm. After inserting the EGFP protein expression cassette between the homologous left arm and the homologous right arm, a new pseudorabies virus attenuated strain is constructed, which has reduced pathogenicity compared to the wild-type PRV-DX. When Balb / c mice are infected with this pseudorabies virus, no pathological clinical symptoms appear, indicating its potential for preparing a pseudorabies vaccine. Brief Description of the Drawings

[0018] Figure 1 Schematic diagram of the transfer vector required for the rescue of the EGFP-PRV recombinant virus;

[0019] Figure 2 Result diagram of the successful rescue of the EGFP-PRV recombinant virus;

[0020] Figure 3 One-step growth curve of PRV-DX and the EGFP-PRV recombinant virus;

[0021] Figure 4 Virulence of PRV-DX and the EGFP-PRV recombinant virus;

[0022] Figure 5 Diagram showing the protective effect of the EGFP-PRV recombinant virus as an antigen on mice. Detailed Embodiments

[0023] The first object of the present invention is to provide a method for reducing the virulence of pseudorabies virus, including inserting an EGFP protein expression cassette upstream of the UL40 gene of pseudorabies virus.

[0024] The present invention does not particularly limit the method of inserting the EGFP protein expression cassette. For example, in the examples, the EGFP protein expression cassette is inserted upstream of the UL40 gene by homologous recombination. More specifically, the UL39 gene is used as the homologous left arm, and 9 bases upstream of UL40 and the UL40 gene are used as the homologous right arm. The EGFP protein expression cassette is inserted between the homologous left arm and the homologous right arm. The EGFP protein expression cassette of the present invention includes CMV, EGFP, and SV40 connected in sequence, and the EGFP protein expression cassette includes the nucleotide sequence shown in SEQ ID No. 1.

[0025] In one embodiment of the present invention, the above genetic engineering modification is carried out based on the porcine pseudorabies virus PRV-DX, and the porcine pseudorabies virus PRV-DX has been deposited. Its deposit number is CGMCC No. 14326 and it has been disclosed in Chinese Patent CN116024182A. In this basic strain, the nucleotide sequence of the homologous left arm LA (UL39) is shown in SEQ ID No. 2.

[0026] In PRV-DX, the nucleotide sequence of the homologous right arm RA (9 bases upstream of UL40 and UL40) is shown in SEQ ID No. 3.

[0027] The present invention also provides an attenuated or avirulent pseudorabies virus mutant strain constructed by using the above method.

[0028] In a preferred embodiment of the present invention, it includes mutating the porcine pseudorabies virus PRV-DX as the basic strain to construct the attenuated or avirulent pseudorabies virus mutant strain; the porcine pseudorabies virus PRV-DX has been deposited, and the deposit number is CGMCC No. 14326. In the embodiment of the present invention, there is no obvious difference in virus proliferation between the constructed EGFP-PRV recombinant virus and the parental strain PRV-DX, and it has no virulence.

[0029] The present invention also provides a production method of the above attenuated or avirulent pseudorabies virus mutant strain, including inserting an EGFP protein expression cassette upstream of the UL40 gene of the pseudorabies virus, and obtaining the attenuated or avirulent pseudorabies virus mutant strain through virus rescue.

[0030] In a preferred embodiment of the present invention, vero cells are used as the host cells during virus rescue. The present invention does not particularly limit the method of virus rescue.

[0031] The present invention also provides the application of the above attenuated or avirulent pseudorabies virus mutant strain in the preparation of a pseudorabies vaccine. The EGFP-PRV recombinant virus constructed in the embodiment of the present invention has a good application prospect for pseudorabies vaccines.

[0032] The present invention also provides a pseudorabies vaccine, which includes the above attenuated or avirulent pseudorabies virus mutant strain as an active ingredient and also includes excipients.

[0033] In order to further illustrate the present invention, the following examples are used to describe in detail a method for reducing the virulence of PRV by gene insertion and its application in the preparation of vaccines provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Construct a recombinant mutant strain EGFP-PRV with an EGFP protein expression cassette inserted upstream of the UL40 gene by homologous recombination.

[0036] As Figure 1 shown, using the UL39 gene sequence (SEQ ID No.2) as the homologous left arm (LA), the UL40 gene sequence (including 9 bases between the two genes, SEQ ID No.3) as the homologous right arm (RA), and the independent expression cassette sequence of EGFP (SEQ ID No.1) in the middle, connect them between EcoRⅠ and BamHⅠ of the pUC18 vector by fusion PCR to obtain the recombinant vector pUC18-UL39-EGFP-UL40.

[0037] Extract the genomic DNA of the wild strain PRV-DX (PRV-gDNA), and transfect 293t cells at a ratio of PRV-gDNA:pUC18-UL39-EGFP-UL40 = 1:3. After transfection for about 60 h, collect the culture supernatant and use it to continue culturing vero cells in a 96-well plate. After 48 h, select the wells with green fluorescence under a fluorescence microscope. Freeze-thaw the vero cells in the 96-well plate once, then collect the supernatant of the fluorescent wells and infect vero cells in a six-well plate. After infection for 2 h, discard the supernatant, wash three times with PBS, and then add a low melting point agarose medium containing 2% serum (1:1 mixing of 2% low melting point agarose and 2×DMEM medium containing 4% serum). After it solidifies, invert the cell culture plate and culture it in an incubator. After 36 h, mark the cytopathic effect (CPE) with green fluorescence under a fluorescence microscope. Pick the above CPE with a sterile 20 μL pipette tip, serially dilute it 10-fold into serum-free DMEM medium, and add it to vero cells in a six-well plate for infection for 2 h. Subsequently, culture it with a low melting point agarose medium according to the above method. Repeat the above experiment until all the CPE in the culture plate emits green fluorescence, then pick the CPE, infect vero cells in a six-well plate, and culture them with a liquid medium containing 2% serum.

[0038] After preserving the cultured virus, extract the genomic DNA (EGFP-PRV-gDNA). Sequencing and identification of the EGFP expression cassette sequence showed that all sequences were as expected and no mutations occurred. The results after rescue were as Figure 2 shown, indicating that the rescue of the EGFP-PRV recombinant virus was successful.

[0039] Example 2

[0040] Determine the one-step growth curve of PRV-DX and the EGFP-PRV recombinant virus, and analyze the effect of the inserted mutation on the proliferation characteristics of PRV-DX.

[0041] Determination of the one-step growth curve of the virus:

[0042] The PK15 cells were evenly seeded in 12-well plates. After the cells grew to confluence, PRV-DX and EGFP-PRV were respectively infected at an moi of 0.01. After 2 hours of infection, the cells were washed three times with sterile PBS, and maintenance medium containing 2% serum was added for culture. Culture supernatants were collected at 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h respectively, and the virus titer was determined by vero cells in 96-well plates.

[0043] The vero cells were evenly seeded in 96-well plates. After the cells grew to confluence, the virus solution to be tested diluted 10-fold successively was added to the vero cells in 96-well plates (100 μL / well), and 8 replicates were made for each dilution. After continuous culture for 72h, the appearance of CPE in each well was observed under a microscope, and the virus titer was calculated by the Reed-Muench method.

[0044] The results are as Figure 3 shown, and there is no obvious difference in virus proliferation between the recombinant virus and the parental strain.

[0045] Example 3

[0046] Virus virulence determination.

[0047] PRV-DX and EGFP-PRV were respectively subcutaneously infected into Balb / c mice (10 mice each) at a lethal dose (10 6 TCID 50 ). The death of the mice was observed and recorded every day, and the observation period was 14 days.

[0048] The results are as Figure 4 shown, indicating that the recombinant virus is non-virulent.

[0049] Example 4

[0050] The immune protection effect of EGFP-PRV on the body was detected through a mouse infection experiment.

[0051] Based on the above results, EGFP-PRV (10 7 TCID 50 ) was used as an antigen component to subcutaneously inoculate Balb / c mice (10 mice each). After 14 days, the mice were infected with the PRV-DX strain at a lethal dose (10 6 TCID 50 ). The death of the mice was observed and recorded every day, and the observation period was 14 days.

[0052] The results are as Figure 5 shown, indicating that the body can be protected against virulent attack after immunization with EGFP-PRV.

[0053] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for reducing the virulence of pseudorabies virus, characterized in that: The method comprises inserting an EGFP protein expression cassette upstream of the UL40 gene of pseudorabies virus.

2. The method according to claim 1, characterized in that: The method comprises taking the UL39 gene as the homologous left arm, taking the 9 bases upstream of UL40 and the UL40 gene as the homologous right arm, and inserting the EGFP protein expression box between the homologous left arm and the homologous right arm.

3. The method according to claim 1 or 2, characterized in that: The EGFP protein expression box comprises CMV, EGFP and SV40 connected in sequence.

4. The method according to claim 3, characterized in that: The EGFP protein expression box includes the nucleotide sequence shown in SEQ ID No.

1.

5. An attenuated or avirulent pseudorabies virus mutant strain constructed by the method according to any one of claims 1 to 4.

6. The attenuated or avirulent pseudorabies virus mutant according to claim 5, characterized in that: The attenuated or avirulent pseudorabies virus mutant strain is constructed by taking the porcine pseudorabies virus PRV-DX as the basic strain and mutating the strain; The porcine pseudorabies virus PRV-DX has been deposited with the deposit number CGMCC No.14326.

7. The method for producing the attenuated or avirulent pseudorabies virus mutant according to claim 5 or 6, characterized in that: The method comprises inserting an EGFP protein expression box upstream of the UL40 gene of the pseudorabies virus, and obtaining the attenuated or avirulent pseudorabies virus mutant strain by virus rescue.

8. The production method according to claim 7, characterized in that: Vero cells were used as host cells for virus rescue.

9. Use of the attenuated or avirulent pseudorabies virus mutant strain according to claim 5 or 6 in the preparation of a pseudorabies vaccine.

10. A pseudorabies vaccine, characterized in that: The invention comprises the attenuated or avirulent pseudorabies virus mutant strain as claimed in claim 5 or 6 as an active ingredient, and further comprises auxiliary materials.

Images