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Method for expressing duck-derived avian influenza virus ns1 protein and application of the method

An avian influenza virus, duck-derived technology, applied in the direction of viruses, applications, viral peptides, etc., can solve the problems of affecting downstream protein purification, low secretion efficiency, and difficulty in breaking walls.

Active Publication Date: 2021-02-23
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein expressed by the animal cell expression system has natural biological activity and function, but the operation is cumbersome and the cost of cultivation is high, so it is not suitable for large-scale cultivation
Although yeast cells are easy to operate and have low culture costs, they are difficult to break the wall and have low secretion efficiency, which easily affects the purification of downstream proteins
The protein expressed in Escherichia coli lacks transcriptional and post-translational processing modifications, and the expressed protein activity is not high or has no activity, so it is limited in use

Method used

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  • Method for expressing duck-derived avian influenza virus ns1 protein and application of the method
  • Method for expressing duck-derived avian influenza virus ns1 protein and application of the method
  • Method for expressing duck-derived avian influenza virus ns1 protein and application of the method

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preparation example Construction

[0028] 2) Preparation of NS1 gene cDNA: the viral RNA extracted in step 1) is reverse transcribed into cDNA by reverse transcription primer sequence 5'-AGCAAAAGCAGG-3';

[0029] 3) PCR amplification of NS1 gene:

[0030] Using FQNS1F-BamHI and FQNS1R-XhoI as primers, using the cDNA obtained in step 2) as a template, PCR amplifies the NS1 gene; wherein, the sequences of the FQNS1F-BamHI and FQNS1R-XhoI are as follows:

[0031] FQNS1F-BamHI: GGATCC ATGGACTCCAACACTGTGTCAAG, where the underlined part is the BamHI restriction enzyme site;

[0032] FQNS1R-XhoI: CTCGAG TCAGTGATGGTGATGGTGATGTTTATCGTCGT CGTCTTTATAATCCTTTGGAGAGAGTGGAGGTC, where the underlined part is the XhoI restriction enzyme site;

[0033] 4) Construction of TOPO clone positive plasmid and codon optimization: After the PCR amplification product obtained in step 3) was purified and recovered, the online software was used to analyze the codon usage bias of the NS1 gene and Tetrahymena, and then the NS1 gene was co...

specific Embodiment

[0060] 1.1 Materials

[0061] The duck-origin avian influenza virus A / duck / Fujian / FQ2 / 2007 (H9N2) strain was isolated from Fujian in 2007 by our laboratory (GenBank accession number JF916709). The method of virus isolation is as follows: collect the trachea, lung, and kidney tissues of sick chickens and homogenize them in a sterilized glass grinder, make a suspension of 100 g / L with PBS, freeze and thaw twice, centrifuge at 2500 × g for 10 min, and take The supernatant was filtered and sterilized through a 0.22 μm filter for later use. Inoculate 10-day-old SPF chicken embryos with the treated tissue suspension through the allantoic cavity, incubate at 37°C with 0.2ml each, remove dead chicken embryos within 24 hours, observe twice a day, and collect aseptically for 48-96 hours The allantoic fluid was continuously passed for 3 generations, and the embryo fluid was aseptically recovered and stored at -30°C for future use. Rabbit anti-A / duck / Fujian / FQ2 / 2007 (H9N2) serum and Esc...

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Abstract

The present invention relates to a method for expressing the NS1 protein of duck-derived avian influenza virus and the application of the method. The method mainly includes the following steps: (1) construction of Tetrahymena expression vector pD5H8-NS1; (2) Tetrahymena recombinant cells Construction and screening of strains; (3) induced expression and identification of NS1 protein; the application of the NS1 protein expression method of duck-derived avian influenza virus is used in the preparation of avian influenza virus differential diagnosis kit and vaccine preparation, the present invention Tetrahymena expression system is selected as the expression system of duck-derived avian influenza virus NS1 protein, which can efficiently and stably express duck-derived avian influenza virus NS1 protein. The transgenic expression vector pD5H8 used in the Tetrahymena cell expression system selected by the present invention contains high The metallothionein gene MTT5 promoter provides an important guarantee for the high-efficiency expression of foreign genes in Tetrahymena.

Description

technical field [0001] The invention relates to a method for expressing duck-origin avian influenza virus NS1 protein and application of the method. Background technique [0002] Avian Influenza (Avian Influenza) is an infection and / or disease syndrome of birds (poultry and wild birds) caused by type A influenza virus. The virus consists of 8 gene segments, the shortest of which is a viral non-structural gene (non-structural gene, NS). During virus replication, the NS gene expresses two important functional proteins, namely the 26kDa NS1 protein and the 14kDa NEP (nuclear export protein, NEP) protein. Among them, the NS1 protein is a multifunctional protein with two typical structural domains, namely the RNA-binding domain (RNA-binding domain, RBD) and the effector binding domain (an effector domain, ED), which determine the ability of AIV to be infected. Key determinants of cellular destructiveness, affecting AIV replication, pathogenicity, virulence, and host range. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N15/44C12N15/66C07K14/11G01N33/569A61K39/145A61P31/16
CPCC07K14/005C12N15/66C12N15/79C12N2760/16122C12N2760/16134C12N2800/22G01N33/56983
Inventor 朱春华刘晓东龚晖池洪树陈翠腾陈珍刘斌琼黄瑜
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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