Recombinant sequence containing H7N9 virus HA gene, recombinant baculovirus and application of virus in vaccine preparation

A technology of recombinant baculovirus and gene, applied in the direction of medical preparations containing active ingredients, applications, antiviral agents, etc., can solve the problems of potential safety hazards, reduction of adverse reactions, and inferior cracking vaccines, etc., to achieve high application value and increase Yield and cost reduction effects

Inactive Publication Date: 2015-06-17
特菲(天津)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the 1970s and 1980s, on the basis of split vaccines, a virion subunit vaccine was developed, which only contained surface antigens and no internal antigens and lipids. The adverse reactions after vaccination were further reduced, but the immunogenic

Method used

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  • Recombinant sequence containing H7N9 virus HA gene, recombinant baculovirus and application of virus in vaccine preparation
  • Recombinant sequence containing H7N9 virus HA gene, recombinant baculovirus and application of virus in vaccine preparation
  • Recombinant sequence containing H7N9 virus HA gene, recombinant baculovirus and application of virus in vaccine preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of the recombinant transposable plasmid pFastBac 1-gp64-HA

[0056] 1. Amplification of gp64-HA

[0057]Check the nucleotide sequence of the H7N9 influenza virus HA protein gene from the NCBI database, connect the signal peptide sequence of the gp64 gene and the transmembrane region sequence of the gp64 gene at the 5' end and the 3' end of the fragment, and then insert The 5' end of the signal peptide sequence is connected to a sequence for binding of BssH II enzyme cleavage (BssH II restriction site sequence), and the 3' end of the transmembrane region sequence of the gp64 gene is connected to a sequence for binding of Xba I enzyme digestion (Xba I I restriction site sequence) to form a full-length 1896bp SP-HA-TM sequence (shown in SEQ ID NO: 1), and then submit the SP-HA-TM sequence to Beijing Huada Gene Co., Ltd. for synthesis. The synthetic product SP-HA-TM gene was used as a template, and gp64-F (shown in SEQ ID NO: 2) and gp64-R (shown in...

Embodiment 2

[0060] Embodiment 2: Obtaining of Bombyx mori recombinant baculovirus Bm-gp64-HA

[0061] The recombinant transposable plasmid pFastBac 1-gp64-HA, which was successfully identified as recombination, was transformed into Escherichia coli DH10Bac competent cells containing the baculovirus shuttle vector Bacmid. Cultured on the LB culture plate, after homologous recombination by transposition (the gp64-HA sequence on pFastBac 1-gp64-HA was inserted into the multi-cloning site of Bacmid by homologous transposition), blue-white screening was carried out, and cultured in the dark Pick the white spot after 48h, and continue to shake the white spot in the LB medium containing tetracycline, kanamycin, gentamycin, X-gal and IPTG, and then use isopropanol to extract the recombinant baculovirus genomic DNA. Using M13 universal primers, gp64-F and gp64-R to identify the insertion of the target gene in the recombinant Bacmid by PCR amplification, the successfully inserted plasmid was named ...

Embodiment 3

[0065] Example 3: Expression of HA protein in silkworm BmN cells

[0066] The recombinant baculovirus Bm-gp64-HA was mixed with 3×10 -6 A dose of pfu / cell was used to infect silkworm BmN cells for virus amplification. After 3 to 5 days of infection, the virus liquid was collected, separated and purified, and 10 μL of the supernatant was added to an equal volume of 2× protein loading buffer (100Mm Tris-HCl, 4% SDS, 0.15% bromophenol blue, 10% glycerol), heated at 100°C for 10 min, took 10 μL of the heated mixture for SDS-PAGE analysis, and performed Western Blot detection experiment, the results showed that the silkworm recombinant baculovirus HA protein has been expressed.

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Abstract

The invention provides a recombinant sequence containing H7N9 virus HA gene, a recombinant baculovirus and application of the virus in vaccine preparation. The recombinant sequence SP-HA-TM includes a gene coding H7N9 influenza virus HA protein, a gp64 signal peptide sequence and a transmembrane region sequence, wherein the signal peptide sequence is connected to the 5' end of HA protein gene, and the gp64 transmembrane region sequence is connected to the 3' end of the HA protein gene. The recombinant baculovirus Bm-gp64-HA is inserted into a donor plasmid by the recombinant nucleotide sequence SP-HA-TM and is subjected to homologous recombination with shuttle vector Bacmid's genome by transposition so as to obtain the recombinant baculovirus genome DNA, then the recombinant baculovirus genome DNA transfects silkworm cells, and packaging is carried out in silkworm cells to obtain the recombinant baculovirus. The virus is purified and a heat source is removed so as to obtain a vaccine for human immunization and prevention of avian influenza infection among people.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant nucleotide sequence containing the HA protein gene of H7N9 influenza virus, an amino acid sequence encoded by the recombinant nucleotide sequence, and a recombinant rod-shaped silkworm constructed by using the recombinant nucleotide sequence. Virus, the preparation method of the silkworm recombinant baculovirus, and the application of the silkworm recombinant baculovirus in the preparation of H7N9 influenza vaccine. Background technique [0002] H7N9 influenza is a new type of avian influenza. It was first discovered in Shanghai and Anhui at the end of March 2013. It is the first new subtype of influenza virus discovered in the world. [0003] For viral diseases, the best way to prevent them is to get vaccinated. Due to the long development cycle of live attenuated vaccines, the currently widely used influenza vaccines in the world are purified multi...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N7/01C12N15/866C07K19/00A61K39/145A61P31/16
Inventor 张耀洲赵恩伟张芳伟刘晓敏
Owner 特菲(天津)生物医药科技有限公司
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