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Construction method and application of recombinant porcine reproductive and respiratory syndrome virus expressing African swine fever virus p30 protein

An African swine fever virus, respiratory syndrome technology, applied in the field of bioengineering, can solve the problems of genetic instability, inability to function, loss of coding sequences, etc.

Pending Publication Date: 2019-12-31
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

But even so, the insertion of foreign genes will gradually lose the coding sequence in the process of passage, so that it cannot perform its function
genetic instability

Method used

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  • Construction method and application of recombinant porcine reproductive and respiratory syndrome virus expressing African swine fever virus p30 protein
  • Construction method and application of recombinant porcine reproductive and respiratory syndrome virus expressing African swine fever virus p30 protein
  • Construction method and application of recombinant porcine reproductive and respiratory syndrome virus expressing African swine fever virus p30 protein

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Embodiment 1

[0030] The construction of the recombinant PRRSV plasmid that embodiment 1 expresses ASFV p30 protein

[0031] According to the nucleotide sequences of GenBank accession numbers EF635006 and MH766894, 6 primers were designed for SOE PCR amplification. The process is as follows: figure 2 As shown, then the infectivity of the obtained chimeric recombinant plasmid was verified by cell transfection experiments, the specific process is as follows:

[0032] 1.1 Primer design

[0033] According to the base sequence of HuN4 on GenBank and the base sequence of the popular type II ASFV genome in my country, SOEPCR primers were designed and named as: HF11559, HR13090, ASFV-p30-F2, ASFV-p30-R1, ASFV-p30- The sequences of F4 and ASFV-p30-R2 / R3 are as follows:

[0034] HF11559: 5-TCATACATCCGAGTTCCTGTT-3 (SEQ ID NO. 1)

[0035] HR13090: 5-GAAATATTGTCATGGCGAGGC-3 (SEQ ID NO. 2)

[0036] ASFV-p30-F2: 5-TCATTGAACCAACTTTAGGCCTGAATTGAAatggattttatttaaatatatc-3 (SEQ ID NO. 3)

[0037] ASFV-p30...

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Abstract

The invention provides a construction method of porcine reproductive and respiratory syndrome virus (PRRSV) recombinant plasmid expressing African swine fever virus (ASFV) p30 protein, and a genetically engineering vaccine constructed based on the recombinant plasmid and a construction method thereof. Indirect immunofluorescence is carried out on rPRRSV-p30 infected wells, it is found that the specific fluorescence of anti-PRRSV N protein and anti-ASFV p30 protein appears in the field of vision. Protein samples of rPRRSV-p30 infected wells are collected and analyzed by Western blot, results show that p30 bands of the expected size are found. It is suggested that the recombinant PRRSV expressing African swine fever virus p30 protein can ensure good, efficient and stable expression of foreign protein ASFV p30 protein. However, some studies show that p30 is an important antigen protein, and can be used in the development of ASF vaccine.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a virus recombinant plasmid and a genetic engineering vaccine, and more specifically relates to a PRRS virus recombinant plasmid capable of expressing African swine fever virus (ASFV) p30 protein and genetic engineering vaccine. Background technique [0002] ASFV is an acute, hemorrhagic and highly contagious infectious disease caused by ASFV infection in domestic pigs or wild boars. It is characterized by a short course of disease, high fever and hemorrhagic lesions, and the mortality rate of acute infection is as high as 100%. Serious threat to the global pig industry. And no effective vaccine or treatment has yet been developed. Since the first ASF epidemic occurred in Shenyang, Liaoning, China in August 2018, it has spread rapidly to more than 20 provinces across the country, causing huge economic losses to China's pig industry. Judging from the overall situation...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/34C12N15/40A61K39/12A61P31/14A61P31/20
CPCC12N15/85C07K14/005A61K39/12A61P31/14A61P31/20C12N2770/10022C12N2770/10034C12N2710/12022C12N2710/12034A61K2039/53A61K2039/552A61K2039/70
Inventor 童光志高飞姜一峰童武李国新郑浩周艳君刘长龙虞凌雪李丽薇
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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