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Establishment of method for preparing recombinant protein vaccine of type A H1N1 influenza virus

A technology of recombinant protein and influenza virus, which is applied in the field of zoonotic disease research, can solve the problems of limited quantity and shortage of vaccines, and achieve the effect of low production cost, low cost and high biological safety

Inactive Publication Date: 2010-06-02
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The nationwide vaccination work is in full swing, but because the vaccines approved by the state for H1N1 are all inactivated viruses, their production needs to amplify the virus in vitro and then inactivate it for the preparation of immunization, and its production is limited in quantity. Great restrictions, vaccines will be in short supply in a short period of time

Method used

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  • Establishment of method for preparing recombinant protein vaccine of type A H1N1 influenza virus
  • Establishment of method for preparing recombinant protein vaccine of type A H1N1 influenza virus
  • Establishment of method for preparing recombinant protein vaccine of type A H1N1 influenza virus

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Construction of Influenza A H1N1 Influenza Virus HA Truncated Protein Gene Prokaryotic Expression Vector

[0024] PUC57-HA truncated protein and vector pGEX-6P-1 were digested with BamHI and SalI, and the HA truncated protein gene and vector pGEX-6P-1 were recovered; then ligated with T4DNA ligase, bathed at 22 degrees for 1 hour, and transformed into Escherichia coli Rossetta. cultured at 37`C, randomly selected a number of single colonies, put them into LB liquid medium and cultured at 37°C for 5 hours, added IPTG to a final concentration of 1mM when the OD value of the bacteria was about 0.6, and identified by small expression , to pick positive clones.

Embodiment 2

[0025] Example 2 A large amount of induced expression and protein purification of H1N1 influenza virus HA

[0026] The pGEX-6P-1-HA truncated protein was expressed in Escherichia coli Rosetta, the clone was inoculated into 3ml LB buffer containing 100ug / ml ampicillin, 37°C, overnight, 220rpm; the overnight culture was diluted 1:100 to 1L containing 100ug / ml ampicillin in LB buffer, 37°C, 220rpm, culture for 5h; add IPTG with a final concentration of 0.1mM, 22°C, 180rpm, continue to culture for 22h; centrifuge at 8000g, 15min, 4°C to collect bacteria; Suspend in 50mM Tris-Cl, 200mM NaCl pH8.0 to 25ml, add 5mg lysozyme, RT, 0.5h; sonicate the solution until the solution is clear, 12000rpm, 20min centrifuge 3 times; use the Glutathione of GE healthcare for the supernatant - Sepharose4B column material purification to obtain purified fusion protein. PSP digests the GST tag, and the column material is purified to obtain the HA truncated protein with the tag removed.

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Abstract

The invention belongs to the field of zoonosis researches, and relates to a method for preparing a vaccine of zoonosis. In the method, an HA gene in a cDNA sequence of a type A H1N1 California virus strain (A / California / 08 / 2009(H1N1)) is selected as a research content; the method comprises the following steps of: firstly, analyzing a sequence of the part of an HA protein leaked outside an envelope according to structural biology software; secondly, selecting a part with high immunogenicity according to an antigenic analysis, and constructing a prokaryotic expression vector pGEX-6P-1-HA by using a genetic fragment obtained through a gene synthesis method; and thirdly, converting a masculine recombinant plasmid into Escherichia coli to obtain a recombinant strain (Escherichia coli BL21 rosseta / pGEX-6P-1-HA truncated protein). Through a plurality of chromatography methods, a purified HA truncated protein of the H1N1 is obtained; and by utilizing the expression vector and an adjuvant together to immune organisms, the protective effect on the H1N1 virus is achieved.

Description

technical field [0001] The invention belongs to the field of zoonosis research and relates to a preparation method of a vaccine for zoonosis. The HA gene in the published A / California / 08 / 2009 (H1N1) cDNA sequence was selected as the research content. First, the sequence of the part of the HA protein leaking outside the envelope was analyzed according to the structural biology software. According to the antigenicity analysis, the part with higher immunogenicity was selected, and the prokaryotic expression vector pGEX-6P-1-HA was constructed by using the gene fragment obtained by the gene synthesis method; the positive recombinant plasmid was transformed into Escherichia coli to obtain the recombinant strain (Escherichia coli BL21rosseta / pGEX-6P-1-HA truncated protein); the purified HA truncated protein of H1N1 was obtained by various chromatographic methods, and the expressed protein was used together with an adjuvant to immunize organisms together to achieve Prophylactic prot...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61P31/16
Inventor 华权高沈鹤霄
Owner CUSABIO TECH LLC
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