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Infectious bronchitis virus rH120-YZS1 delta 5a as well as construction method and application thereof

A technology for rh120-yzs1 and bronchitis, which is applied in the fields of molecular biology and genetic engineering, and can solve problems such as unclear functions

Active Publication Date: 2020-03-24
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both 3ab and 5ab are virus-encoded nonstructural proteins that can be detected in IBV-infected cells, but their functions are still unclear
What they have in common is that the genome of the infectious bronchitis virus H120 vaccine strain or the Beaudette strain genome is used as the backbone, and the S or S1 gene of the isolate is used to replace the corresponding region. genotype S or S1 gene

Method used

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  • Infectious bronchitis virus rH120-YZS1 delta 5a as well as construction method and application thereof
  • Infectious bronchitis virus rH120-YZS1 delta 5a as well as construction method and application thereof
  • Infectious bronchitis virus rH120-YZS1 delta 5a as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Construction of recombinant plasmid pMDTM9-YZS1Δ5a

[0071] Preparation of biological material:

[0072] The preservation number of chicken infectious bronchitis virus isolate IBYZ is CGMCC No.14682. JM109 genetically engineered bacteria were purchased from Dalian Bao Biological Engineering Co., Ltd.; pMD18-T vector was purchased from Dalian Bao Biological Engineering Co., Ltd.

[0073] 1. Cloning of gene fragment S1 of extramembrane region of spike protein of IBYZ strain

[0074] Primers were designed based on the whole genome sequence of chicken infectious bronchitis virus isolate IBYZ to amplify the gene fragment S1 of the extramembrane region of the spike protein. The Bsa I restriction endonuclease recognition sequence was introduced at the 5' end of the amplification primer pair; the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Using the genomic RNA of chicken infectious bronchitis virus isolate IBYZ as a template, high...

Embodiment 2

[0086] Construction of full-length cDNA of rH120-YZS1Δ5a

[0087] Preparation of biological material:

[0088] Recombinant plasmids pMDTM1, pMDTM2, pMDTM3, pMDTM4, pMDTM5, pMDTM6, pMDTM7, pMDTM8 and pMDTM10 carrying genome fragments of the H120 vaccine strain of infectious bronchitis virus [Zhou Sheng, Dai Yabin, Tang Mengjun et al. Chicken infectious bronchitis virus H120 vaccine strain whole Construction of long cDNA infectious clones. China Poultry, 2010, 32(23):22-26.], constructed and preserved by the Poultry Disease Prevention and Control Laboratory of Jiangsu Institute of Poultry Science. The applicant commits to release to the public for 20 years from the filing date.

[0089] JM109 carrying recombinant plasmids pMDTM1-pMDTM8, pMDTM9-YZS1Δ5a and pMDTM10 were inoculated into LB medium respectively, and the plasmids were extracted using TaKaRa MiniBEST Plasmid Purification Kit. After the extracted plasmid was digested with BsaI, the TM1, TM2, TM3, TM4, TM5, TM6, TM7, T...

Embodiment 3

[0100] Identification of biological characteristics of rH120-YZS1Δ5a strain

[0101] Preparation of biological material:

[0102] Molecular clone rH120 of chicken infectious bronchitis virus H120 vaccine strain was constructed and preserved by Jiangsu Institute of Poultry Science. The applicant commits to release to the public for 20 years from the filing date. SPF chicken embryos were purchased from Beijing Meria Verton Experimental Animal Technology Co., Ltd.

[0103] 1. Dwarfing test of chick embryos

[0104] The P3 seed virus of the rH120-YZS1Δ5a strain was diluted 1:100 times with PBS, inoculated into the allantoic cavity of five 11-day-old SPF chicken embryos, 0.2mL / piece, and incubated at 37°C for 144h, and the chickens were observed Embryo changes. The results showed that chick embryos died regularly after rH120-YZS1Δ5a strain infected chicken embryos, showing characteristic changes: thickened amniotic membrane, clinging to the embryo body, and the embryo body was ...

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Abstract

The invention provides an infectious bronchitis virus rH120-YZS1 delta 5a as well as a construction method and application thereof, and belongs to the technical field of molecular biology and gene engineering. The infectious bronchitis virus rH120-YZS1 delta 5a is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.10499. The infectious bronchitis virus rH120-YZS1 delta 5a can be adopted to express S1 protein of QX type and Mass type infectious bronchitis viruses at the same time, so that the infectious bronchitis virus rH120-YZS1 delta5a can be used for preparing bivalent vaccines for preventing infectious bronchitis.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to an infectious bronchitis virus rH120-YZS1Δ5a and its construction method and application. Background technique [0002] Chicken Infectious Bronchitis Virus (IBV) is a representative species of Coronaviridae Coronaviridae. Its genome is a single-stranded positive-sense RNA, about 27.6kb in length, and has at least 10 obvious open reading frames (ORF), which encode the virus respectively. The structural and non-structural proteins of the ORF have been determined to be located in the order of 5'lab-S-3ab-E-M-5ab-N-3'. Both 3ab and 5ab are virus-encoded non-structural proteins that can be detected in IBV-infected cells, but their functions are still unclear. Studies have shown that the deletion of the 3ab and 5ab coding regions does not affect the replication of IBV and the pathogenicity of the virus [Casais R, Davies M, Cavanagh D, et ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/86C12N15/66C07K14/165A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/552A61K2039/70A61P31/14C07K14/005C12N7/00C12N15/66C12N15/86C12N2770/20021C12N2770/20022C12N2770/20034C12N2770/20043
Inventor 周生唐梦君程旭姜逸俞燕赵秀美高明艳韦玉勇
Owner JIANGSU INST OF POULTRY SCI
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