Large-scale preparation method of H5N1 avian influenza virus-like particle vaccines

An avian influenza virus and virus-like technology, applied in the field of genetic engineering, can solve the problem that the production steps of virus-like particles are not suitable for large-scale production, and achieve the effect of good development and application prospects.

Inactive Publication Date: 2011-01-19
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to the implementation steps of the patent, it is not possible to start from influenza virus to produce virus-like particle vaccine pr...

Method used

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  • Large-scale preparation method of H5N1 avian influenza virus-like particle vaccines
  • Large-scale preparation method of H5N1 avian influenza virus-like particle vaccines
  • Large-scale preparation method of H5N1 avian influenza virus-like particle vaccines

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Experimental program
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Embodiment 1H5

[0027] Construction and detection of embodiment 1H5N1 avian influenza virus virus-like particles (VLPs) vaccine

[0028] 1. Design and synthesis of primers

[0029] H5N1 Human Avian Influenza Virus Vaccine Strains Recommended by WHO

[0030] Hemagglutinin HA coding sequence (Genbank No.GQ454861), neuraminidase NA sequence (Genbank No.GQ454862) and matrix protein M sequence (Genbank No.GQ454867), respectively designed and synthesized three pairs of specific primers to amplify HA, NA and M1 genes respectively; designed a general primer for type A influenza virus for cDNA synthesis; each primer Synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.; the sequences and numbers of each primer are as follows:

[0031] 1) HA gene PCR amplification primers

[0032] Upstream primer P1 (SEQ ID NO.1)

[0033] 5′-TACCCGGGGCCAGCATGGAGAAAATAGT-3′

[0034] Downstream primer P2 (SEQ ID NO.2)

[0035] 5′-GGCTAGCTTAAATGCAAATTCTGC-3′

[0036] (The 5' end of the amplification ...

Embodiment 2H5

[0128] Example 2 Scale preparation and verification of H5N1 avian influenza virus virus-like particles (VLPs) vaccine

[0129] 1. Optimization of Sf9 insect cell culture conditions and large-scale cell suspension culture

[0130] 1) Optimum inoculum concentration optimization of recombinant baculovirus

[0131] Take well-growing Sf9 cells, digest them with 0.25% trypsin, inoculate them into 6-well plates, the cells form about 80% monolayer, and inoculate them at different dilutions of MOI=0.01, 0.1, 1, 3, 5 and 10 respectively The third-generation recombinant baculovirus, after adsorption for 1 hour, was replaced with fresh Grace's culture medium (2% fetal bovine serum), and cultured statically at 27°C. The virus fluid was harvested every day.

[0132] According to the method in step 8.2, measure the virus titration of each MOI infection dose after different propagation time, and determine the optimal infection dose and proliferation culture time of the recombinant baculov...

Embodiment 3H5

[0151] Functional detection of embodiment 3H5N1 avian influenza virus virus-like particles (VLPs) vaccine

[0152] In order to evaluate the safety and immune efficacy of the H5N1 avian influenza virus VLPs vaccine, the present invention uses BALB / c mice as animal models.

[0153] 1. Safety evaluation of H5N1 avian influenza virus VLPs vaccine

[0154] Get 20 healthy female BALB / c mice aged 5-6 weeks and divide them into 2 groups at random, 10 in each group. The mice in group A were intramuscularly injected with H5N1 avian influenza virus VLPs vaccine (10 μg / 100 μl per mouse), and the mice in group B Group mice were intramuscularly injected with 100 μl sterile PBS as a negative control.

[0155] The mice were observed for 21 days after injection, and the weight change was measured. On days 5, 7, 9, 14 and 21, blood was collected from the tail and the number of white blood cells was counted.

[0156] The results showed that there was no significant difference in body weight ...

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Abstract

The invention provides a large-scale preparation method of new H5N1 avian influenza virus-like particle vaccines, which sequentially comprises the following steps: (1) constructing and extracting the recombinant plasmid rbacmid/HANAM1; (2) transfecting the recombinant plasmid rbacmid/HANAM1 to Sf9 insect cells to revive the recombinant baculovirus, and assembling virus-like particles in the Sf9 cells; and (3) culturing and recombining the Sf9 cells in a large scale, and purifying the virus-infected particles and the virus-like particles. The method is suitable for large-scale preparation of human anti-highly pathogenic H5N1 avian influenza VLPs vaccines, and has the advantages of safety, high efficiency and good development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a construction and large-scale preparation method of novel highly pathogenic H5N1 avian influenza virus-like particles (VLPs). technical background [0002] Influenza is an important infectious disease that endangers human health. The pathogen belongs to the Orthomyxoviridae family Influenza virus, which is divided into three types: A, B and C according to the antigenicity of the virus nucleoprotein. Influenza A Virus (IAV) is an enveloped RNA virus, and its genome consists of 8 segmented single-stranded negative-sense RNA segments. According to the antigenicity of hemagglutinin (HA) and neuraminidase (NA) on the surface of virions, influenza A viruses are divided into 16 different HA subtypes (H1-H16) and 9 different NA subtypes (N1 -N9). [0003] Influenza A virus has a wide host range and high variability, and is considered to be the greatest threat to future influ...

Claims

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Application Information

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IPC IPC(8): A61K39/145C12N7/01A61P31/16C12R1/93
Inventor 朱丹丹陶攀璩骁潘兹书
Owner WUHAN UNIV
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