Method for constructing encephalomyocarditis virus infections clone

A technology of encephalomyocarditis virus and infectious cloning, applied in the field of genetic engineering, can solve problems such as limitations in molecular virology research, achieve strong controllability, increase success rate, and ensure fidelity

Inactive Publication Date: 2009-03-18
CHINA AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] EMCV is a member of the genus Myovirus in the Picornaviridae family. Its genome is a single-stranded positive-strand RNA that does not form a DNA inter...

Method used

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  • Method for constructing encephalomyocarditis virus infections clone
  • Method for constructing encephalomyocarditis virus infections clone
  • Method for constructing encephalomyocarditis virus infections clone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Transformation of low copy plasmid vector

[0025] According to the complete sequence of the low copy plasmid vector pWSK29 and Instructions for MultiSite-directed Mutagenesis Kit (Stratagene, Denmark), design 1 pair of palindromic primers Spe-Upper and Spe-Lower to mutate the Spe I site at position 3536, and design 1 pair of palindromic primers BssH-Upper and BssH-Lower The BssH II site at position 5124 was mutated. Design primers based on a nonsense sequence of the PRRSV Nsp2 gene, add BssH II and Cla I restriction sites to the 5' end of the upstream primer pNSP2-F, and sequentially add the 5' end of the downstream primer pNSP2-R BssH II and Sph I two enzyme cutting sites, with HS DNA Polymerase (Takara, Dalian) amplified this fragment and digested it with BssHII. At the same time, the mutated pWSK29 vector was digested with BssHII, and the digested product was dephosphorylated, and then T4 ligase (Promega, USA ) Ligate the above-mentioned amplifie...

Embodiment 2

[0029] Example 2 Extraction of encephalomyocarditis virus total RNA

[0030] The F8 generation cells of porcine encephalomyocarditis virus BJC3 were used to culture the virus, and the total RNA of the BJC3 virus was extracted according to the specific steps in the kit manual using the QIAampViral RNA Mini Kit (Qiagen, Germany). The obtained RNA was dissolved in 20 μL RNase-free water (Ambion Inc., USA), and frozen at -70°C.

Embodiment 3

[0031] Example 3 RT-PCR method for segmental amplification of the full-length cDNA of encephalomyocarditis virus

[0032] According to the whole genome sequence of porcine encephalomyocarditis virus BJC3 (GenBank No. DQ464062), design 3 pairs of primers covering the whole genome of the virus, the primer sequences and their positions in the BJC3 genome are as shown in Table 2: the upstream primers for full-length amplification T7 RNA polymerase promoter core sequence is added to T7-SegA-F to ensure high abundance and high quality RNA during in vitro transcription; Cla I restriction site is added to T7-SegA-F and ClaI-SegB-F ; Sph I and BamH I two enzyme cutting sites are added to SegC-R; Point mutations are introduced into primers SegB-R and SegC-F, and after amplification, T at position 6262 of the genome is mutated to A, and the codon after mutation Both CGA and the pre-mutation codon CGT encode arginine, which is a synonymous mutation. The obtained sequence GAATTC is the rec...

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Abstract

The invention relates to a method for constructing infectious clone of an encephalomyocarditis virus. The method comprises the following steps: extracting the total RNA of the encephalomyocarditis virus; piecewise amplifying the full-length cDNA of the encephalomyocarditis virus by an RT-PCR method, and separately constructing sub-clones; and directionally cloning each fragment of the cDNA sequentially to a low copy plasmid carrier by a double-enzyme cutting method so as to obtain the infectious clone of the encephalomyocarditis virus. The method has small operation difficulty and strong controllability, and can obtain stable infectious clone of the encephalomyocarditis virus, thereby providing an effective tool for the molecular virology research of the encephalomyocarditis virus and the development of vaccines.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing infectious clones of encephalomyocarditis virus. Background technique [0002] Encephalomyocarditis is an acute infectious disease characterized by encephalitis, myocarditis or perimyocarditis in pigs, some mammals and even primates, and is caused by encephalomyocarditis virus (EMCV). EMCV has a wide range of hosts and has been found in various rodents, wild animals and primates. Humans can also be infected, but most of them do not show any symptoms. Pigs are the most widely and severely infected animals. In addition to causing fatal myocarditis and encephalitis in piglets, they can also cause reproductive disorders in sows. [0003] At present, there is no effective treatment drug and vaccine for porcine encephalomyocarditis in China, and it is mainly prevented by comprehensive control measures. Because EMCV has a close relatio...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/63
Inventor 杨汉春盖新娜郭鑫张国庆朱书
Owner CHINA AGRI UNIV
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