High fidelity porcine reproductive and respiratory syndrome virus attenuated strain and application thereof

A technology for respiratory syndrome and pig reproduction, applied in the directions of viruses, antiviral agents, virus antigen components, etc., can solve the problems of easy mutation of vaccine strains, easy loss of immunogenicity, poor genetic stability, etc., to reduce production costs and safety. The effect of increasing sexuality and reducing tropism

Active Publication Date: 2017-08-18
YICHUN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PRRS attenuated vaccine strains are easy to mutate and have poor genetic stability, resulting in easy loss of immunogenicity and easy return of virulence

Method used

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  • High fidelity porcine reproductive and respiratory syndrome virus attenuated strain and application thereof
  • High fidelity porcine reproductive and respiratory syndrome virus attenuated strain and application thereof
  • High fidelity porcine reproductive and respiratory syndrome virus attenuated strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 PRRSV NJ strain is attenuated by passage on Marc-145 cells

[0019] 1. Cell Culture

[0020] Marc-145 cells were grown in cell culture medium at 37 °C, 5% CO 2 cultured in an incubator. Marc-145 cells were grown into a monolayer and used to inoculate the virus. Cell culture medium: DMEM cell culture medium containing 10% (volume percent concentration) inactivated (56° C. for 30 min) calf serum and double antibodies (penicillin and streptomycin, both at a concentration of 100 U / mL).

[0021] 2. Cultivation of PRRSV NJ strain

[0022] PRRSV NJ-a strain is a virulent American type of PRRSV (disclosed in Zheng Qisheng, Li Peng, Cao Ruibing, Hou Jibo, Chen Puyan, etc.) isolated from an acutely ill pig herd in Nanjing. Construction of vaccinia virus Ankara strain and its biological characteristics, Acta Biological Engineering, 2008,24(5):766-773). In the present invention, the PRRSV NJ-a strain disclosed in "Construction and biological characteristics of a reco...

Embodiment 2

[0025] Embodiment 2 High-fidelity PRRSV attenuated strain cultivation and identification in vitro

[0026] 1. Continuous passage of PRRSV seed poison chemical mutagenesis

[0027] Continuous passage of mutagenesis: culture Marc-145 cells to 70% confluence with cell culture medium (composition is the same as in Example 1), and then add mutagen Ribavirin (ribavirin, purchased from sigma company) with a final concentration of 200 μM for pretreatment for 2 h , remove the culture medium, add the F80 generation of PRRSV NJ strain according to the MOI of 1 (PRRSV NJ strain seed virus is obtained after 80 generations of Marc-145 cells, the virus is an attenuated strain), infect for 1h, and wash twice with PBS buffer ; Then, add cell culture solution (composition is the same as in Example 1) containing 200 μM mutagen to continue culturing, and finally harvest the virus. The virus was continuously passaged for 20 generations according to the above method.

[0028] At the same time, a ...

Embodiment 3

[0058] Example 3 High-fidelity PRRSV attenuated strain tissue tropism identification

[0059] Inoculate PAM (porcine alveolar macrophages) and Marc-145 cells (MOI=1) with the seed virus of PRRSV-NJRb strain, respectively, and pass 5 times in succession, observe the changes of virus CPE (cytopathic effect) in each generation, and harvest different generations The titer of the secondary virus was determined. At the same time, the F80 generation of PRRSV NJ strain was subcultured 5 times in the same way as the control group. The results showed that no CPE was seen in the PAM cells inoculated with the first-generation virus NJRb P1 of the PRRSV-NJRb strain (referring to the virus obtained after one generation of the seed virus), while typical CPE appeared in the control group ( figure 2 ), PRRSV-NJRb strain after each generation also all do not see CPE to occur (results not shown). The results of virus titer determination are shown in Table 4. The results showed that compared ...

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Abstract

Belonging to the field of biotechnology, the invention provides a high fidelity porcine reproductive and respiratory syndrome (PRRS) virus attenuated strain and application thereof. The attenuated strain is porcine reproductive and respiratory syndrome virus PRRSV-NJRb strain with a microbial preservation number of CGMCC No.13800. The invention also provides a preparation method of an attenuated strain virus solution, and the method includes: using Marc-145 cell to proliferate the high fidelity porcine reproductive and respiratory syndrome virus attenuated strain, performing cell lysis, conducting centrifugation and taking the supernatant liquid, thus obtaining the virus solution. The invention also provides a porcine reproductive and respiratory syndrome attenuated vaccine, the active component of which is the porcine reproductive and respiratory syndrome virus attenuated strain. The attenuated strain has low mutation rate and high genetic stability, can still maintain the original immunogenicity and virulence after 100 passages, and has good protection on high pathogenic PRRS. The attenuated vaccine provided by the invention has good protection on high pathogenic PRRS, and has higher safety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-fidelity attenuated strain of porcine reproductive and respiratory syndrome virus and its application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS), also known as PRRS, is an important severe contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), which has caused serious damage to the pig industry in my country. huge economic losses. PRRS inactivated vaccines are basically ineffective, and attenuated PRRS vaccines have the advantages of fast immune protection and high efficacy, and are currently the only means of prevention and control of PRRS in my country. However, PRRS attenuated vaccine strains are easy to mutate and have poor genetic stability, resulting in easy loss of immunogenicity and easy return of virulence. Contents of the invention [0003] The purpose of the present in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N7/00A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552C12N7/00C12N2770/10021C12N2770/10034
Inventor 李鹏成侯继波郑其升张晓燕陈瑾于小明乔绪稳张元鹏
Owner YICHUN UNIVERSITY
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