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Infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes

A technology for respiratory syndrome and infectious cloning, applied in the field of bioengineering, to achieve the effect of simple and convenient method, good market prospect and huge economic value

Inactive Publication Date: 2020-09-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Simultaneously, aiming at the actual problem of lacking safe and cross-protected PRRS vaccines at present, the infectious cloning platform of HP-PRRSV natural attenuated strains of the present invention can also be used to develop safer and more efficient novel PRRSV genetically engineered vaccines

Method used

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  • Infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes
  • Infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes
  • Infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1—Porcine reproductive and respiratory syndrome virus strong and weak strains XJ17-5 and JSTZ1712-12 genomic full-length cDNA clone construction and rescue

[0032] 1.1 pACYC177-New vector construction

[0033] Referring to the previously reported infectious clone construction method and improving on the original basis, a method for constructing the XJ17-5 and JSTZ1712-12 infectious clone platform was designed. Use DNAMAN software to compare and analyze the whole genome sequences of XJ17-5 and JSTZ1712-12, and select four single restriction sites of PacⅠ, AflⅡ, AscⅠ and NotⅠ for the full length of the gene when constructing the XJ17-5 and JSTZ1712-12 infectious cloning platform segment insertion. Such as figure 1 As shown in the schematic diagram of vector transformation, the low-copy plasmid pACYC177 was selected as the original vector, and a vector containing the CMV promoter, four corresponding restriction sites (PacⅠ, AflⅡ, AscⅠ and NotⅠ) and bovine growth...

Embodiment 2

[0054] Example 2—Construction and rescue of porcine reproductive and respiratory syndrome virus infectious clones rXJ17-5-EGFP and rJSTZ1712-12-RFP strains containing specific fluorescent markers

[0055] 1.1 Plasmid construction

[0056] In order to conveniently determine the rescue situation of infectious cloned virus and analyze the replication of rescued virus, this study inserted enhanced green fluorescent protein (Enhancedgreen fluorescent protein, EGFP) and red Fluorescent protein (Red fluorescent protein, RFP). Such as Image 6 As shown, a gene fragment with a full length of 1100bp and containing 4 restriction sites (AscI, KpnI, BclI and BglII) and EGFP was synthesized by Suzhou Jinweizhi Company and inserted into the pUC57 vector, named pAscI-KpnI - EGFP-BclI-BglII. A pair of primers (RFP-F-KpnⅠ: 5'-ATTGAAGGTACCGCCACCATGGCCTCCTCCGAGGA-3' and RFP-R-BclⅠ: 5'-TGCCGCGGAATGATCACTACAGGAACAGGTGGTGGC-3') designed according to the pDsRed-Express-C1 plasmid kept in the labor...

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Abstract

The invention relates to infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes, and belongsto the technical field of bioengineering. The two constructed infectious clone viruses are an XJ17-5 virulent strain and a JSTZ1712-12 natural weak strain which are highly homologous but are significantly different in virulence based on an HP-PRRSV genome. A method for constructing infectious clone is simple and convenient, DNA transfection is directly performed, in vitro transcription into virusRNA and then transfection are not needed, and the shortcomings of being not stable in RNA in vitro and easy in degradation are overcome. The obtained HP-PRRSV infectious clone platform can be appliedto research on an in vitro virus reproduction mechanism and an infective mechanism. The constructed HP-PRRSV natural weak strain infectious clone virus can be used for researching a safer and more efficient novel PRRSV genetic engineering vaccine, and prevention and control of Chinese PRRSV is facilitated.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the construction, rescue, transformation and application of infectious clones of two strong and weak strains of porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an animal infectious disease caused by PRRS virus (PRRSV), characterized by reproductive disorders in sows and respiratory symptoms in pigs of all ages, commonly known as "pig blue ear disease". . In 1987, the disease was first reported in the United States, and then spread all over the world, seriously endangering the development of pig industry. In 1995, PRRSV was isolated for the first time in my country. In 2006, "pig high fever" broke out in my country, causing high fever (≥41°C), 50-100% morbidity and 20-100% mortality, which caused huge economic losses to my country's pig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/85C12N15/40C12N15/65A61K39/12A61P31/14
CPCC12N7/00C12N15/85C12N15/65A61P31/14C12N2770/10021C12N2770/10022C12N2770/10034
Inventor 陈南华叶梦雪朱建中
Owner YANGZHOU UNIV
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