Chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming influence of maternal antibody of Newcastle disease and construction method thereof

A technology of Newcastle disease virus and maternal antibody is applied in the field of developing Newcastle disease vector vaccine, which can solve the problems of changing antigenicity and reducing virus replication efficiency, and achieve the effect of broad application prospect and high reproduction performance.

Inactive Publication Date: 2017-10-17
YANGZHOU UNIV
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Whether only replacing the extracellular regions of NDV F and HN proteins with functionally similar regions of different viruses will reduce the replication efficiency of the virus and change the antigenicity, has not yet been studied in China

Method used

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  • Chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming influence of maternal antibody of Newcastle disease and construction method thereof
  • Chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming influence of maternal antibody of Newcastle disease and construction method thereof
  • Chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming influence of maternal antibody of Newcastle disease and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The construction model of recombinant plasmid pNDV / rAI4-T4FHN is as follows figure 1 .

[0032] Construction Step 1: Modification of Newcastle Disease Virus AI4 Genome

[0033] Biomaterial preparation:

[0034] 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. pCR2.1 vector: purchased from Invitrogen; AMV reverse transcriptase, High fidelity DNA polymerase, T4 DNA ligase, and Agarose Gel DNA Extraction Kit were purchased from Roche; transfection reagent SuperFect and plasmid extraction kit (QIAprep Spin MiniPrep Kit) It is the product of QIAGEN; the rest of the conventional reagents are domestic analytical grade.

[0035]...

Embodiment 2

[0073] Embodiment 2: the biological characteristic identification of recombinant virus

[0074] 1. Determination of MDT and ICPI

[0075] Chicken embryo mean death time (mean death time, MDT) measurement: Recombinant virus rAI4-gB was made 10 times (10 times) respectively with sterilized physiological saline. -6 、10 -7 ...10 -10 ) gradient dilution, each dilution was inoculated with 5 9-11-day-old SPF chicken embryos, 0.1 mL per embryo, and 5 chicken embryos inoculated with normal saline were set as a control. Incubate at 37°C, discard chicken embryos that died within 24 hours, then observe every 12 hours until 120 hours, and calculate the MDT of the virus according to the OIE standard method. Results: Chicken embryos inoculated with 5 dilutions of the virus did not die within 120 hours, and the MDT value of this strain was greater than 120 hours.

[0076] Determination of intracerebral pathogenicity index (ICPI) inoculation in chicks: take fresh virus allantoic fluid with...

Embodiment 3

[0079] Embodiment 3: Immunological potency test of vaccine carrier strain

[0080] The 1-day-old commercial chickens and SPF chickens were randomly divided into 10 chickens / group, and kept in isolation, and were inoculated with the vaccine vector strain AI4-T4FHN and LaSota attenuated vaccine respectively through nasal drops and eye drops, for 10 days. 6 EID 50 / only, and the PBS inoculated group was set as the control. Venous blood was collected from each test chicken 2 weeks after immunization, and the level of seroconversion against the immune strain was detected. The result is as image 3 , AI4-T4FHN and LaSota immunized chickens did not show any clinical symptoms, indicating that the AI4-T4FHN vaccine vector strain is not pathogenic to chickens and can be used safely. LaSota attenuated vaccine immunization of 1-day-old commercial chickens is greatly affected by maternal antibodies, which cannot effectively stimulate antibody production and can only maintain serum titer...

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Abstract

The invention belongs to the technical field of application of reverse genetics and gene replacement, and particularly relates to a chimeric Newcastle disease virus vaccine vector candidate strain capable of overcoming the influence of a maternal antibody of a Newcastle disease and a construction method of the chimeric Newcastle disease virus vaccine vector candidate strain. The candidate strain is avian paramyxovirus type-1 AI4-T4FHN, and the preservation number is CGMCC No:12987. An established reverse genetics manipulation platform for a Newcastle disease virus gene type-VII attenuated virus strain AI4 is utilized to replace the corresponding part of a genome of the strain AI4 through the sequence of an extracellular region of envelope proteins F and HN of an avian paramyxovirus type-2, and the recombinant virus AI4-T4FHN is obtained through saving. The cross reaction capacity of the avian paramyxovirus type-2 and the Newcastle disease virus are very weak, and the avian paramyxovirus type-2 is not pathogenic for a chicken. The chimeric virus has a high reproductive capacity similar to that of a female parent virus, and meanwhile, the chimeric virus is effectively replicated in the chicken body with the maternal antibody of the Newcastle disease at high level, and is a novel vaccine carrier.

Description

technical field [0001] The invention relates to the application of reverse genetics and gene replacement technology to rescue a chimeric Newcastle disease virus vaccine vector candidate strain AI4-T4FHN which overcomes the influence of chicken Newcastle disease maternal antibody, and is used for developing Newcastle disease vector vaccine. 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 refe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/45C12R1/93
CPCC12N7/00C12N15/85C12N2760/18121C12N2760/18152
Inventor 刘秀梵刘晶晶王晓泉刘晓文胡顺林
Owner YANGZHOU UNIV
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