Duck source coronavirus M protein gene and cloning method and application thereof

A technology of coronavirus and gene, applied in the field of duck-derived coronavirus M protein gene and its cloning

Inactive Publication Date: 2010-12-15
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, people's research on the IBV M protein gene is still in the exploratory stage

Method used

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  • Duck source coronavirus M protein gene and cloning method and application thereof
  • Duck source coronavirus M protein gene and cloning method and application thereof
  • Duck source coronavirus M protein gene and cloning method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] The cloning method of the M gene of embodiment 1 duck origin coronavirus ZZ2004

[0024] (1) PCR primer design Design the following primer sequences:

[0025] Upstream primer 5'-gtcgctagaggagaatggta-3';

[0026] Downstream primer 5'-atacgcttccgtgctctta-3'.

[0027] (2) The enrichment method of ZZ2004 strain

[0028] Dilute the allantoic fluid infected by ZZ2004 strain 1:10 with sterilized PBS, inoculate 0.2ml into the chorioallantoic cavity of 9-day-old SPF chicken embryos, incubate at 37°C for 36-48h, and place it at 4°C for 12h Harvest chick embryo allantoic fluid.

[0029] Take 50ml of SPF chick embryo allantoic fluid infected by ZZ2004 strain respectively in 3 tubes of sterilized centrifuge tubes, centrifuge at 5000rpm for 20min; discard the precipitate respectively, take the supernatant in another sterilized centrifuge tube, centrifuge at 12000rpm for 20min; For precipitation, take the supernatant and add it to a 50ml ultracentrifuge tube, make up the deficienc...

Embodiment 2ZZ200

[0076] Example 2ZZ2004 Strain M Gene Deduced Amino Acid Sequence Homology Alignment

[0077] Using DNAstar software to compare the deduced amino acid sequence of the M gene of the ZZ2004 strain with the seven reference strains registered in GenBank, the results showed that the homology rates between the deduced amino acid sequence of the M gene of the ZZ2004 strain and the reference strain were 93.9%-91.1%, respectively; Compared with the classical strain Beaudette strain, the M protein of ZZ2004 strain mainly concentrated in two regions of 3-16aa and 211-222aa, and some scattered point mutations still existed in other regions; compared with the SAIBK strain with the highest homology rate, The mutation section is mainly concentrated in the 3-17aa region, and there are some scattered point mutations in other regions, see figure 1 .

[0078] Amino acid sequence genetic phylogenetic tree analysis of ZZ2004 strain M protein (see figure 2 ) results showed that the isolate ZZ2004...

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Abstract

The invention relates to a duck source coronavirus M protein gene. The gene has a nucleotide sequence and an amino acid sequence shown in SEQ ID No.3 and 4; and the cloning method comprises the following steps: selecting a duck source coronavirus ZZ2004 strain, adopting 5'-GTCGCTAGAGGAGAATGGTA-3' as an upstream primer and adopting 5'-ATACGCTTCCGTGCTCTTA-3' as a downstream primer; and amplifying a duck source coronavirus membrane protein gene full-length fragment by adopting an RT-PCR method, and then connecting the fragment with a pMD18-T carrier. Compared with similar coronaviruses, the ZZ2004 has larger degree of variation and wider inflection spectrum and is important for studying the membrane protein gene structure, revealing the prevalence and variation rules in a molecular biology level, and having important significance on developing effective genetic engineering subunit protein vaccine and medicines for preventing and curing IB so as to prevent and control the occurrence and prevalence of the IB.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a duck-derived coronavirus M protein gene and its cloning method and application. Background technique [0002] In 1937, coronaviruses (Coronaviruses) were first isolated from chickens. In 1965, the first human coronavirus was isolated. It is named "coronavirus" because it can be observed under an electron microscope that there are obvious rod-shaped particle protrusions on its outer membrane, making it look like the crown of a medieval European emperor. According to the serological characteristics of the virus and the differences in nucleotide sequences, the Coronaviridae family is currently divided into two genera, coronavirus and torovirus. The representative strain of Coronaviridae is Avian infectious bronchitis virus (IBV). The virus is an enveloped, moderately pleomorphic spherical or oval virion with rod-shaped protrusions on the surface. [0003] Avian infectious bronchi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N15/10C07K14/165A61K39/215A61P31/14A61P11/00
Inventor 姚四新刘兴友赵淑秋刘金华李学斌王丽荣王宪文王自良赵坤张兆沛
Owner HENAN INST OF SCI & TECH
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