Avian influenza virus hemagglutinin antigen, CHO cell strain expressing the same, preparation method, and vaccine

An avian influenza virus and hemagglutinin technology, which is applied in the biological field, can solve the problems of unreachable poison price, long cycle, and excessive production of waste, and achieve the effects of good antigen immunogenicity, low production cost and high immune efficacy.

Active Publication Date: 2019-01-01
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, inactivated vaccines for avian influenza are produced using chicken embryos or MDCK whole suspension cells. The vaccine production process using chicken embryos is complicated, and the chicken embryos used may be contaminated by exogenous viruses. It is necessary to inoculate chicken embryos with venom, collect allantoic fluid, and inactivate , the collection of allantoin liquid chicken embryo waste requires high-pressure treatment, which produces more production waste and brings pressure on environmental protection
The production of avian influenza vaccine by MDCK full-suspension cells requires the process of acclimating the seed virus to adapt to the cells, which takes a long period and sometimes fails to reach the expected poison price. The cultivation cost and production process are relatively complicated

Method used

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  • Avian influenza virus hemagglutinin antigen, CHO cell strain expressing the same, preparation method, and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 A sequence expressing hemagglutinin protein

[0057] Download the HA gene sequence of the H5 subtype of avian influenza that is currently circulating and circulating in the past 5 years from Genebank for comparison and analysis, and select the dominant epitope as the component of the vaccine antigen. According to the preference of CHO cell codons, the avian influenza virus The sequence of HA gene is optimized and modified to improve the level of HA protein expressed by the target protein. At the same time, in order to make the HA gene have a broad spectrum of antigenicity, the relatively conservative HA1 epitope antigen sequence was screened, and the 3'end of HA2 was connected to the matrix protein M2e (containing 23 amino acids) in the form of flexible amino acids, forming a piece of artificial The base sequence of the polypeptide, through protein homology modeling, uses the structure and sequence homology to search for protein modules in the PDB library, simula...

Embodiment 2

[0058] Example 2 A recombinant vector expressing hemagglutinin protein

[0059] The above synthesized fragment encoding the amino acid sequence of the HA protein and having a His tag at the C terminal was inserted into the eukaryotic transfer vector pcDNA3.1 through Sal I and Xho I sites. The ligation product was obtained after ligation with T4DNA ligase overnight at 16°C, which was transformed by E. coli competent DH5a and spread on an LB plate containing ampicillin. After culturing overnight at 37°C, the positive colonies were picked and placed on LB containing ampicillin. Cultivate in the medium and extract the plasmid.

Embodiment 3

[0060] Example 3 Identification of restriction digestion of recombinant vector expressing hemagglutinin protein

[0061] Prepare plasmid DNA, select Sal I and Xho I sites for restriction endonuclease digestion, and subject the digested product to 1% agarose gel electrophoresis. The result is as follows figure 1 Shown. Among them, lane M: DNA Marker, lanes 1 to 3: plasmid after digestion, and lane 4: plasmid before digestion. It can be seen from the figure that the target of the expected size appears, indicating that the target gene is successfully inserted into the vector.

[0062] The plasmid with the positive result was sent to Shanghai Shenggong for sequencing and verification, and finally a positive recombinant expression plasmid was obtained. Its sequence is shown in SEQ ID NO.1, among which the 1402-1431, 1441-1476 and 1486-1512 epitopes are Between the flexible amino acids encoded by CCTCCCAGC are connected.

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Abstract

The invention provides an avian influenza virus hemagglutinin antigen, a CHO cell strain expressing the same, a preparation method, and a vaccine, and relates to the field of biotechnology. The avianinfluenza virus hemagglutinin antigen is a hemagglutinin protein expressed by a sequence shown in SEQ ID NO. 1 and has the advantage of broad spectrum as an antigen, and the hemagglutinin protein is aprotein expressed by a eukaryotic cell, so that a conformational epitope which loses the antigen is avoided. The preparation method of the avian influenza virus hemagglutinin antigen and the cell strain expressing the avian influenza virus hemagglutinin antigen can obtain a hemagglutinin protein having broad spectrum and antigen immunogenicity. The vaccine of the avian influenza virus hemagglutinin antigen has low production cost and high immunological efficiency. After immunizing SPF chickens which are 3 to 4 weeks old, an H5 subtype antigen can be detected on the 14th day to determine the geometric mean titer (GMT) of an HI antibody (GMT) to be not less than 1:64, which is superior to conventional vaccines.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to an avian influenza virus hemagglutinin antigen and a CHO cell strain expressing it, a preparation method and a vaccine. Background technique [0002] Avian influenza virus (AIV) is a single-stranded negative-stranded virus, spherical or filamentous, with a spherical diameter of 80-120nm. The gene component consists of 8 segments, which encode 10 proteins. Among them, HA, NA and M are located on the surface of the virus's envelope and are the main protective antigens of AIV. Located under the double lipid envelope of the virus is the matrix protein M, which is the most abundant protein in the virus particle, which forms the framework of the virus envelope. Two glycoprotein spikes encoded by viral genes are embedded in the lipid bilayer membrane, namely hemagglutinnin (HA) and neuraminidase (NA), both of which are subtypes of influenza viruses And its antigenicity is very easy to mutate. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N5/10C12N15/85A61K39/145A61P31/16
CPCA61K39/12A61K2039/552A61P31/16C07K14/005C12N5/0682C12N15/85C12N2510/02C12N2760/16122C12N2760/16134
Inventor 贺笋李俊辉于新茂李延涛寇春张伟
Owner 天康生物制药有限公司
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