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Chimeric Newcastle disease virus vector H7 (avian influenza) live vaccine candidate strain capable of overcoming effect of Newcastle disease virus maternal antibody of chick and a construction method thereof

A technology of Newcastle disease virus and maternal antibody, which is applied in the field of chimeric Newcastle disease virus vector H7 live vaccine candidate strain and its construction to overcome the influence of chick Newcastle disease maternal antibody, and can solve problems such as limiting the application of NDV vector vaccine

Inactive Publication Date: 2017-11-24
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This greatly limits the application of NDV vector vaccines in commercial chicken flocks

Method used

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  • Chimeric Newcastle disease virus vector H7 (avian influenza) live vaccine candidate strain capable of overcoming effect of Newcastle disease virus maternal antibody of chick and a construction method thereof
  • Chimeric Newcastle disease virus vector H7 (avian influenza) live vaccine candidate strain capable of overcoming effect of Newcastle disease virus maternal antibody of chick and a construction method thereof
  • Chimeric Newcastle disease virus vector H7 (avian influenza) live vaccine candidate strain capable of overcoming effect of Newcastle disease virus maternal antibody of chick and a construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of recombinant plasmid pNDV / rAI4-T4FHN

[0034] Construction Step 1: Modification of Newcastle Disease Virus AI4 Genome

[0035] Biomaterial preparation:

[0036] The full-length expression vector pNDV / rAI4 of NDV AI4 strain (Hu Z, Hu S, Meng C, et al. Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs. Avian Diseases 2011,55(3):1759- 1769) was constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University.

[0037] pCR2.1 vector: purchased from Invitrogen; AMV reverse transcriptase, High fidelity DNApolymerase, T4DNA ligase, and Agarose Gel DNA Extraction Kit were purchased from Roche; transfection reagent SuperFect and plasmid extraction kit (QIAprep Spin MiniPrep Kit) were The products of QIAGEN; the rest of the conventional reagents were of domestic analytical grade.

[0038] Design mutati...

Embodiment 2

[0077] The construction model of the recombinant plasmid pNDV / rAI4-T4FHN-H7 is as follows figure 1 .

[0078] Construction step 1: Construction of full-length expression clones expressing HA protein of avian influenza H7N9 subtype strain using chimeric Newcastle disease virus AI4-T4FHN as a vector

[0079] Biomaterial preparation:

[0080] A / Chicken / Jiangsu / JX148 / 2014 (JX148), the H9N2 subtype epidemic strain of avian influenza virus, was isolated and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University. GISAID database serial number is EPI841962

[0081] pCR2.1 vector: purchased from Invitrogen; Dephospharylation (BAP) Kit: purchased from Bao Biological Engineering (Dalian) Co., Ltd.; AMV reverse transcriptase, High fidelity DNA polymerase, T4 DNA ligase, AgaroseGel DNA Extraction Kit were purchased from Roche; Transfection reagent SuperFect and plasmid extraction kit (QIAprepSpin MiniPrep Kit) are ...

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Abstract

The invention relates to a chimeric Newcastle disease virus vector H7 (avian influenza) live vaccine candidate strain capable of overcoming an effect of a Newcastle disease virus maternal antibody of a chick and a construction method thereof. The preservation number of the Newcastle disease vaccine candidate strain is CGMCC No.13798. The construction method includes: making use of a reverse genetic manipulation platform of a chimeric Newcastle disease virus rAI4-TFHN which is capable of being replicated in the existence of a Newcastle disease antibody, inserting an HA (hemagglutinin) sequence of a subtype H7N9 (avian influenza) virus strain into a full-length transcription vector of an AI4-T4FHN strain genome so as to obtain a full-length cDNA (complementary deoxyribonucleic acid) clone pNDV / rAI4-T4FHN-H7 of a recombinant Newcastle disease virus genome containing the subtype HA gene of the H7N9 (avian influenza) virus. The recombinant virus AI4-T4FHN-H7 obtained through transfection is high in propagation titer in chick embryos, still capable of expressing HA protein stably after continuous passage and applicable to large-scale production of vaccine and can be used for manufacturing the vaccine.

Description

technical field [0001] The invention relates to the application of reverse genetic technology to rescue a recombinant vaccine strain rAI4-T4FHN-H7 expressing the HA protein of avian influenza H7 subtype virus by using chimeric Newcastle disease virus as a carrier, and is used for vaccine development. Background technique [0002] Reverse genetics manipulation technique (Reverse genetics manipulation technique) refers to the reverse transcription of RNA virus genomic RNA into cDNA, various in vitro artificial manipulations are performed on it at the DNA molecular level, and new viral genome cDNA and various auxiliary proteins are assembled. A research technique for RNA viruses [Neumann G, Whitt M A, Kawaoka Y. Adecade after the generation of a negative-sense RNA virus from cloned cDNA–what have we learned? Journal of General Virology, 2002, 83(11): 2635-2662.], also known as full-length infectious cDNA cloning technology, and often referred to as "virus rescue". Since the "r...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C12R1/93
CPCC12N7/00C12N15/85C12N2760/16122C12N2760/18121
Inventor 刘秀梵刘晶晶胡顺林王晓泉
Owner YANGZHOU UNIV
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