Vaccine composition, preparation method and application thereof

A vaccine composition and Newcastle disease technology, applied in the field of vaccine composition, can solve the problems such as the decline in the prevention and control ability of Newcastle disease gene type VII epidemic strains

Active Publication Date: 2015-06-24
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prevalence and spread of gene type Ⅶ strains are becoming more and more common and have caused real losses to farms. Vaccination is one of the main measures to prevent the disease. The use of vaccines and inactivated vaccines can still play a certain role in

Method used

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  • Vaccine composition, preparation method and application thereof
  • Vaccine composition, preparation method and application thereof
  • Vaccine composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Construction and detection of H9N2 subtype avian influenza virus-like particles (VLPs)

[0025] 1. The design and synthesis of primers are based on the hemagglutinin HA coding sequence and neuraminidase NA sequence in the genome sequence of the avian influenza virus SZ strain preserved by our company (the preservation number is CCTCC V201240, and the preservation unit is the China Center for Type Culture Collection). and matrix protein M sequence, 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 was synthesized by Shanghai Bioengineering Technology Co., Ltd. The sequences of the primers are as follows:

[0026] 1) HA gene PCR amplification primers

[0027] Upstream primer AIV-P1:

[0028] 5'-AGGATCCATGGAAACAATATCACTAATAAC-3'

[0029] Downstream primer AIV-P2:

[0030] 5'-AGGTA-CCTTATATACAAATGTTGCATCTGC-3' ...

Embodiment 2

[0094] Example 2: Construction and detection of genotype VII Newcastle disease virus-like particles (VLPs)

[0095] 1. Design and synthesis of primers Three pairs of specific primers were designed and synthesized according to the sequences of the F gene, HN gene and M gene of the Newcastle disease gene type VII strain in GenBank (GenBank: AF431744.3) to amplify F and HN respectively and M gene; each primer was synthesized by Shanghai Bioengineering Technology Co., Ltd. The sequence and numbering of each primer are as follows:

[0096] Amplification primers for F gene:

[0097] NDV-P1: 5'-CGTCGACTGCTTATAGTTAGTTC-3'

[0098] NDV-P2: 5'-TGCTCTTTGGTTGCTTGTTCCCAG-3'

[0099] Amplification primers for HN gene:

[0100] NDV-P3: 5'-GCAGCCTGTGTGTCAATTCCGAT-3'

[0101] NDV-P4: 5'-CTACCCGTGTTCTCCCTTGTTG-3'

[0102] Amplification primers for M gene:

[0103] NDV-P5: 5'-AGGACAATCGGGCTGTACTTT-3'

[0104] NDV-P6: 5'-CAGGTGAACTAACTATAAGCAGTC-3'

[0105] 2. Total RNA extraction and cDN...

Embodiment 3

[0132] Example 3: Large-scale preparation and verification of avian influenza and Newcastle disease dual virus-like particles

[0133] 1. Optimization of SF9 insect cell culture conditions and large-scale cell suspension culture

[0134] 1) Optimum inoculum concentration optimization of recombinant baculovirus

[0135] 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 The third-generation recombinant baculovirus was adsorbed for 1 hour, replaced with fresh Grace's culture medium (2% fetal bovine serum), and cultured statically at 27°C. Harvest the virus fluid.

[0136] Determination of virus titer: take well-grown Sf9 cells, digest with 0.25% trypsin, add Grace’s culture medium to suspend cells, and dilute the concentration to 5×10 5 Cells / ml, inoculate 6-well plates at 1ml / well, and culture at 27°C for 48-72h.

[0137...

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Abstract

The invention provides a vaccine composition, which comprises influenza virus-like particles and new castle disease virus-like particles. According to the invention, bird flu virus-like particles and new castle disease virus-like particles are prepared to obtain the vaccine composition, compared with chick embryo-produced vaccine compositions, The production of the vaccine composition can employ a large-volume bioreactor technology and large scale culture of insect cells to prepare antigen, when output is ensured, product quality stability and homogeneity are guaranteed, no live virus is related during the production process, and no hidden trouble on biological safety aspect is generated.

Description

technical field [0001] The present invention relates to a vaccine composition, specifically, the present invention relates to a vaccine composition containing avian influenza virus-like particle antigen and Newcastle disease virus particle antigen, as well as its preparation method and application. Background technique [0002] Virus-like particles (VLPs) are hollow particles between 15nm and 400nm assembled from structural proteins of viruses. The principle of VLPs preparation is to highly express one (or several) structural proteins of a certain virus in vitro, so that they can automatically assemble into hollow particles similar in shape to natural viruses. The method is mainly to clone viral structural protein genes into expression vectors, and then transfer these vectors into prokaryotic or eukaryotic cells for expression. [0003] Avian influenza (Avian influenza, AI) is the abbreviation of avian influenza. It is an acute respiratory infectious disease caused by avian...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61K39/17A61P31/16A61P31/14
Inventor 张许科孙进忠白朝勇田克恭
Owner PU LIKE BIO ENG
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