表达基因Ⅶ型新城疫病毒F蛋白重组LaSota疫苗株的构建

发布时间：2018-07-29 11:11

【摘要】：基因Ⅶ型新城疫病毒(NDV)为我国NDV的主要流行病毒株,而经典疫苗株LaSota不能对鸡群起到有效的免疫保护。为研发针对NDV基因Ⅶ型流行病毒株的疫苗,本研究采用反向遗传操作技术,将LaSota的F基因编码区(CDS)替换为基因Ⅶ型流行病毒株CK/CH/LHLJ/1/06株的F基因CDS,并将替换后的F蛋白的裂解位点突变为LaSota的裂解位点,构建重组质粒pNDFLsp-mF。将pNDFLsp-mF与表达NP、P和L蛋白的辅助质粒共转染BSR-T7/5细胞,拯救获得表达基因Ⅶ型NDV F蛋白的重组病毒(rLaSota-mF)。生长曲线结果显示,与LaSota相似,rLaSota-mF能够在鸡胚中有效复制,病毒滴度EID50为109.375/0.1 mL。生物学特性测定结果显示rLaSota-mF的MDT为144.6 h,表明其为低毒力病毒株。将拯救获得的病毒连续传15代后所替换的基因及突变的位点能够在r LaSota-mF中稳定存在。本研究为研究与开发针对NDV不同基因型流行病毒株的疫苗奠定基础。
[Abstract]:The gene VII type Newcastle disease virus (NDV) is the main epidemic virus strain of NDV in China, and the classic vaccine strain LaSota can not provide effective immune protection to the chickens. In order to develop a vaccine against the NDV gene VII virus strain, the reverse genetic manipulation technique is used to replace the F gene encoding region of LaSota (CDS) as a gene VII virus. The F gene CDS of the strain CK/CH/LHLJ/1/06 strain and the splitting site of the replacement F protein were mutated to the cracking site of LaSota, and the recombinant plasmid pNDFLsp-mF. was constructed to co transfect pNDFLsp-mF with NP, P and L proteins to co transfect BSR-T7/5 cells, and to save the recombinant virus of the expressed gene type VII NDV protein. The results showed that, similar to LaSota, rLaSota-mF could be effectively replicated in the chicken embryo, the virus titer EID50 was 109.375/0.1 mL. biological characteristics and the MDT of rLaSota-mF was 144.6 h, indicating that it was a low virulence virus strain. This study laid the foundation for research and development of vaccines for different genotypes of NDV.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽呼吸道病创新团队;山东省寿光市畜牧兽医局;
【基金】：国家公益性行业(农业)科研专项(201303033) 国家自然科学基金(31502088)
【分类号】：S852.65

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