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Newcastle disease chimeric virus marker vaccine strain as well as construction method and application thereof

A technology for Newcastle disease virus and chimeric virus, applied in the field of Newcastle disease chimeric virus marker vaccine strain and its construction, can solve the problems of limited protective power and no protection of epidemic strains, and achieve high reproductive titer and good immunogenicity. , the effect of excellent growth characteristics

Active Publication Date: 2020-08-25
ZHEJIANG VBIOSCI INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country's existing vaccines have limited protection against epidemic strains, or even basically no protection

Method used

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  • Newcastle disease chimeric virus marker vaccine strain as well as construction method and application thereof
  • Newcastle disease chimeric virus marker vaccine strain as well as construction method and application thereof
  • Newcastle disease chimeric virus marker vaccine strain as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of full-length cDNA clone of recombinant viral genome

[0033] The main experimental material medium is MEM medium containing 10% fetal bovine serum, NDV vaccine strain La Sota was purchased from China Veterinary Drug Administration, BHK-21 cells were purchased from ATCC, SPF chicken embryos and SPF chicken were purchased from Boehringerville Provided by the Experimental Animal Center.

[0034] Main reagents Plasmid extraction kit and gel recovery kit were purchased from Tiangen Company; restriction endonuclease and T4 DNA ligase were purchased from NEB Company; virus RNA extraction kit was purchased from Tiangen Company; E.coli DH5α competent cells Purchased from the whole gold biology; pMDl8-T Vector and GXL DNA Polymerase was purchased from TaKaRa; Lipofectamine TM 2000 transfection reagent was purchased from Invitrogen.

[0035] 1. Primer design and synthesis According to the synthetic sequence of chicken Newcastle disease gene type VII and the sequ...

Embodiment 2

[0050] Construction of helper plasmids

[0051] According to the full-length genome sequence of La Sota strain, primers were designed to amplify the open reading frames (ORFs) encoding nucleoprotein NP, phosphoprotein P and polymerase protein L, respectively. The primers are shown in Table 3. The three ORFs were respectively cloned into the downstream of the CMV promoter of the eukaryotic expression vector pCI-neo. The constructed helper plasmids were named pCI-NP, pCI-P and pCI-L, respectively.

[0052] The construction primer sequence of table 3 auxiliary plasmid

[0053]

[0054] Note: The underlined part is the restriction site.

Embodiment 3

[0056] Construction of BHK T7 cells expressing T7 RNA polymerase

[0057] (1) Synthesize the sequence based on the T7 RNA polymerase sequence (SEQ ID: FJ881694.1) recorded on the NCBI official website to construct the PMV-T7 RNA polymerase plasmid; (2) Ligate the PMV-T7 RNA polymerase plasmid with the P1020 vector overnight, The ligation product was transformed into DH5α competent cells, and the P1020-T7 RNA polymerase positive plasmid was screened out by PCR identification and sequencing identification; (3) The recombinantly constructed P1020-T7 RNA polymerase plasmid was used in the plasmid extraction kit of Tiangen Biochemical Technology Co., Ltd. Extract the plasmid and perform follow-up experiments after confirmation; (4) Transfect the plasmid into cultured BHK-21 adherent cells based on electroporation; Cultivate the cell line, and complete the screening process of the cell line; (6) amplify the screened cell line, and use WB detection to screen the positive cell line wi...

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Abstract

The invention relates to a Newcastle disease chimeric virus marker vaccine strain as well as a construction method and application thereof, and belongs to the field of rescue and application of Newcastle disease chimeric vaccine strains. A Newcastle disease virus reverse genetic operation platform is utilized to mutate an F protein cleavage site of a Newcastle disease gene GVII type strain into acleavage site of an attenuated strain, F and HN genes of a mutated gene VII type Newcastle disease strain and NP, P, M and L of a gene II type NDV La Sota strain construct a full-length chimeric cDNAsequence, and a 18bp nucleotide marker sequence is inserted into a non-coding region between P and M. A Newcastle disease chimeric virus NDV DC strain is obtained through transfection cell rescue. Theconstructed chimeric virus can reach relatively high culture titer in chick embryos and cells. The chimeric strain contains envelope surface glycoprotein of the gene VII type Newcastle disease strain, contains a skeleton of the gene II type strain, and has immunogenicity of the Newcastle disease gene VII type strain and high reproduction and high safety characteristics of the gene II type La Sotastrain.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a marker vaccine strain of Newcastle disease chimeric virus and its construction method and application. Background technique [0002] Chicken Newcastle disease, also known as Asian chicken plague or pseudo-fowl plague, is an acute septicemia infectious disease caused by Newcastle disease virus, which is highly contagious. All chickens can be infected, and domestic chickens are the most susceptible group, and the susceptibility of young chickens is higher than that of adult chickens. The mortality rate of the disease is relatively high, and the onset has no obvious seasonal characteristics, and the disease can be infected throughout the year, but spring and autumn are the high incidence seasons of the disease. [0003] Affected chickens have typical symptoms of dyspnea, diarrhea, neurasthenia, and mucosal and serosal hemorrhage. At the beginning of the on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/295A61K39/17A61P31/14C12Q1/70C12R1/93
CPCC12N7/00C12N15/85A61K39/12A61P31/14C12Q1/701C12N2760/18121C12N2760/18152C12N2760/18134C12N2800/107A61K2039/5252A61K2039/552A61K2039/70
Inventor 张凌云闫召璐谢田田张博郑杰马宁宁
Owner ZHEJIANG VBIOSCI INC
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