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Construction method and application of HVT co-expressing NDV HN and IBDV VP2 genes

A co-expression, gene technology, applied in the field of animal biological products, can solve problems such as increasing the cost of vaccines

Active Publication Date: 2017-08-29
北京邦卓生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The attenuated type I CVI988 / Rispens strain has a good protective effect on MD, but as a strictly cell-associated virus, it must be stored and transported in liquid nitrogen, which greatly increases the cost of the vaccine

Method used

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  • Construction method and application of HVT co-expressing NDV HN and IBDV VP2 genes
  • Construction method and application of HVT co-expressing NDV HN and IBDV VP2 genes
  • Construction method and application of HVT co-expressing NDV HN and IBDV VP2 genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] ——Construction of recombinant virus rHVT-HN

[0038] 1. Construction of recombinant plasmid PHVT-DS

[0039] First construct a recombinant plasmid for homologous recombination, which contains homologous sequences on both sides of the US2 gene, and primers (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8);

[0040] HVT-US2 left sense: 5-GATgaattcA CATCGGGCCA CGTTCCGCC-3 29 (SEQ ID NO: 5);

[0041] HVT-US2 left antisense: 5-ATAGTCGACt taattaaGAT GAGCTGACGT GTGGAAT-337 (SEQ ID NO: 6).

[0042] HVT-US2 right sense: 5-ATCgtcgacA CTAATATGGG CACACCCAC-3 29 (SEQ ID NO: 7);

[0043] HVT-US2 right antisense: 5-ATCaagcttT GGCCCATCTA GGTGATTAT-3 29 (SEQ ID NO: 8).

[0044] The HVT genome was used as a template for amplification, and the amplified left and right arm sequences were cloned into PUC18 through restriction sites. The cloned recombinant plasmid containing the left and right arms was named PHVT-DS.

[0045] 2. Construction of PHVT-DS-NDV-HN

[0046] Accordin...

Embodiment 2

[0052] ——Construction of recombinant turkey herpesvirus rHVT-HN-VP2

[0053] The recombinant virus rHVT-HN was used as a carrier platform to express the VP2 gene of chicken infectious bursal virus (IBDV). The specific construction method is as follows:

[0054] Extract the HVT virus genomic DNA according to conventional methods, according to the full gene sequence of the HVT-FC126 strain (GenBank: AF291866.1) published in Genebank, design primers (sequence 9 and sequence 10) to amplify the left homology arm and connect to PMD19-Tsimple Vector, constructed into plasmid pMD19-L. Primers (Sequence 11 and Sequence 12) were designed to amplify the promoter (Sequence 2), and the promoter was cloned into PMD19-L to construct plasmid PMD19-L-PRO. Then design primers (SEQ ID NO: 13 and SEQ ID NO: 14) to amplify the VP2 gene (SEQ ID NO: 3), clone the VP2 gene into PMD19-L-PRO, and construct the plasmid PMD19-L-PRO-VP2. Design primers (sequence 15 and sequence 16) to amplify PolyA (se...

Embodiment 3

[0070] ——Validation of Recombinant Viruses

[0071] 1. rHVT-HN recombinant virus

[0072] 50PFU of rHVT-HN and HVT were inoculated on 12-well cell culture plates. After obvious plaques appeared in culture, pour out the growth medium, fix with cold acetone:ethanol (3:2) fixative solution for 10 min, wash once with PBS, add 0.5 mL (1:100 Diluted) anti-NDV single-factor serum, placed in a constant temperature incubator at 37°C for 1 hour, washed 3 times with PBS, added 0.5mL FITC-labeled anti-IgG fluorescent antibody to each well, placed in a constant temperature biochemical incubator at 37°C, and reacted for 1 hour, After washing with PBS for 3 times, observed under an inverted fluorescence microscope, the plaques showed specific green fluorescence, while the CEF cells in the control group had no fluorescence after HVT infection. The results indicated that the NDV-HN gene could be well and correctly expressed.

[0073] 2. rHVT-HN-VP2 recombinant virus

[0074] Inoculate 50PFU...

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Abstract

The invention relates to a construction method of a recombinant herpesvirus of turkey rHVT-HN strain expressing Newcastle disease virus (HN) gene. The recombinant virus can be used for preparing vector vaccines to prevent Marek's disease and Newcastle disease. The invention further relates to a construction method and application of a recombinant herpesvirus of turkey co-expressing the Newcastle disease HN gene and infectious bursa fabricius VP2 gene. The recombinant herpesvirus of turkey rHVT-HN strain is a recombinant virus obtained by recombining an expression box of the Newcastle disease HN gene and the infectious bursa fabricius VP2 gene into the HVT. The recombinant virus can serve as a production virus seed to be used for preparing virus live vector vaccines which can prevent both the Marek's disease and the Newcastle disease as well as infectious bursa fabricius.

Description

technical field [0001] The invention relates to the construction and application of an HVT co-expressing NDV HN and IBDV VP2 genes. Belonging to the field of animal biological products, Background technique [0002] Chicken Marek's disease (Marek`s Disease, MD) is the most common lymphoproliferative disease of chickens caused by chicken Marek's disease virus (Marek`s Disease Virus, MDV), which belongs to the B group of cell-associated herpesviruses. It is characterized by lymphoid cell infiltration, hyperplasia, and tumor formation in peripheral nerves, iris, skin, muscles, and various internal organs. [0003] According to the three serotypes of MDV, the current commercial MDV vaccines are divided into attenuated type I CVI988 / Rispens strain, non-pathogenic type II SB1 strain and type III Herpesvirus of turkey (HVT) FC126 strain . The attenuated type I CVI988 / Rispens strain has a good protective effect on MD, but as a strictly cell-associated virus, it must be stored and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/295A61K39/245A61K39/17A61K39/12A61P31/22A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70C12N7/00C12N2710/16321C12N2710/16334C12N2720/10034C12N2760/18134
Inventor 李延鹏皮向成
Owner 北京邦卓生物科技有限公司
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