Porcine reproductive and respiratory syndrome virus as well as cloning vector and gene insertion method thereof

A technology for respiratory syndrome and cloning vectors, applied in the direction of viruses/bacteriophages, microorganism-based methods, biochemical equipment and methods, etc., which can solve the problems that mutant clones are difficult to rescue

Inactive Publication Date: 2020-11-27
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insertion of foreign genes between overlapping sequences will cause some amino acid mutations in adjacent coding regions, making it difficult for mutant clones to be rescued

Method used

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  • Porcine reproductive and respiratory syndrome virus as well as cloning vector and gene insertion method thereof
  • Porcine reproductive and respiratory syndrome virus as well as cloning vector and gene insertion method thereof
  • Porcine reproductive and respiratory syndrome virus as well as cloning vector and gene insertion method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The construction of the full-length infectious clone (pGXAM) of embodiment 1 Gxnn1396-P96 strain PRRSV ( figure 1 )

[0055] Viral RNA was extracted from Gxnn1396-P96 virus-infected cell cultures, and using this RNA as a template, avian myeloblastic disease virus (AMV) reverse transcriptase (TaKaRa, Dalian, China) and anchored poly (T) Reverse transcription was performed with primer Qst for 1 h to obtain the first-strand cDNA. The obtained cDNA was used as a template, and 4 cDNA fragments covering the whole genome were amplified with PfuUltra high-fidelity DNA polymerase (Stratagene) and 4 pairs of primers (SEQ.NO.ID.1-8 in the sequence table), named respectively as PRRSV -A, PRRSV-B, PRRSV-C, PRRSV-D, then cloned into Zero In the vector, 4 intermediate plasmids were obtained. The PRRSV-A containing the 5' end to 2379 nucleotides and the 5' end Pac I sequence was constructed. The PRRSV-B containing the 2108-7728 nucleotide sequence was constructed. The PRRSV-C con...

Embodiment 2

[0056] Embodiment 2 allows the construction of the recombinant infectious clone pGX45BSTRS of exogenous gene insertion ( image 3 )

[0057]Using the forward primer GF11706 (SEQ.NO.ID.10 of the sequence listing) and the reverse primer 45BSR (SEQ.NO.ID.11 of the sequence listing), an upstream fragment was amplified from the trunk of the pGXAM infectious clone, which contained BstB I and sbfI restriction enzyme sites. Using the pGXAM infectious clone as a template, a downstream fragment was amplified with forward primer 45BSF (sequence listing SEQ.NO.ID.12) and reverse primer JX14402MluIR (sequence listing SEQ.NO.ID.13). Contains BstBI and SbfI restriction endonuclease sites. The obtained upstream and downstream fragments are used as the template for the second round of fusion PCR, and GF11706 (sequence listing SEQ.NO.ID.10) and JX14402MluIR (sequence listing SEQ.NO.ID.13) are used as primers to amplify the obtained After the product is purified and recovered, it is cloned in...

Embodiment 3

[0058] Embodiment 3 Construction of PRRSV recombinant infectious clone pGX45BSTRS-GFP expressing marker gene GFP between ORF4 and ORF5a ( image 3 )

[0059] Use primer GFP BstBI F (sequence listing SEQ.NO.ID.15) and GFP SbfI R (sequence listing SEQ.NO.ID.16) to amplify the green fluorescent gene GFP with two restriction sites of BstBI and SbfI, After recovering the target fragment, perform double digestion with BstB I and SbfI restriction endonucleases, purify and recover the digested product, connect to the similar restriction region of pGX45BSTRS, and clone the expression marker gene GFP between ORF4 and ORF5a PRRSV recombinant infectious clone pGX45BSTRS-GFP.

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Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus with a preservation number of CCTCC NO:V 202020. Accordingly, the inventor constructs virus genome full-length infectiousclone pGXAM and establishes a corresponding construction method and a gene insertion method. According to a vector, Gxnn1396-P96 is used as a female parent, and Gxnn1396-P96 is used as a male parent,a PRRSV full-length infectious clone vector is constructed by means of molecular biology, an independent transcription regulating sequence (TRS) is inserted into ORF4 and ORF5a on the basis of cDNA cloning, and the PRRSV recombinant virus for expressing a green fluorescent protein (GFP) gene is constructed. The successful construction of the fluorescence labeled virus lays a foundation for the development of recombinant porcine reproductive and respiratory syndrome virus genetic engineering vaccines.

Description

technical field [0001] The invention belongs to the technical field of porcine reproductive and respiratory syndrome virus, and in particular relates to a strain of porcine reproductive and respiratory syndrome virus, a cloning vector thereof and a gene insertion method thereof. Background technique [0002] Porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV) is one of the important infectious disease pathogens threatening the pig industry in the world, and the porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS) caused by it is highly exposed Infectious diseases mainly cause serious reproductive disorders such as abortion, stillbirth, mummified fetuses, weak piglets, and clinical manifestations such as respiratory disorders, growth stagnation, and high mortality in piglets. PRRSV has brought huge economic losses to the world's swine industry. [0003] PRRSV, a member...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/63C12N15/66A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/552A61P31/14C12N7/00C12N15/63C12N15/66C12N2770/10021C12N2770/10034
Inventor 韦祖樟王玉旭贺微李清清谢欣覃念王豪林思远黄加滨陈樱欧阳康黄伟坚
Owner GUANGXI UNIV
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