仙台病毒BB1株6个编码基因的克隆及表达  

Cloning and Expression of the Six Coding Genes of Sendai Virus BB1 Strain

作　　者：张海凤[1] 杨宇[2] 董小岩[1,3] 吴小兵[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [2]中国检验检疫科学研究院,北京100123 [3]北京五加和分子医学研究所有限公司,北京100176

出　　处：《病毒学报》2009年第3期213-219,共7页Chinese Journal of Virology

基　　金：中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室自主课题;973课题资助(2004CB518800)

摘　　要：在仙台病毒BB1株全基因组序列测定的基础上,用反转录和PCR方法获得了核蛋白基因(N),磷蛋白基因(P),神经血凝素基因(HN),基质蛋白基因(M)、融合蛋白基因(F)和聚合酶蛋白基因(L)等6个编码基因全长克隆;测序结果表明,其序列与Genbank中登录的序列(DQ219803)完全一致。为了提供仙台病毒基因组载体拯救和包装所需的反式作用蛋白,将N、P、M、F、HN和L分别克隆到腺病毒穿梭表达载体pDC316上,将它们分别与腺病毒基因组质粒pBHGlox△E1,3Cre共转染HEK293细胞,获得了6种复制缺陷性重组腺病毒Ad5-N、Ad5-P、Ad5-M、Ad5-F、Ad5-HN和Ad5-L。酶切结果表明6种重组腺病毒穿梭质粒构建正确;用PCR方法证明所获得的6种重组腺病毒分别携带了上述6个编码基因;用重组腺病毒感染LLC-MK2细胞后用Western blotting和免疫荧光方法检测到了相应仙台病毒编码基因的表达。本研究为仙台病毒BB1株全长基因组的拼接和病毒载体包装系统组建打下了基础。Six genes for nucleoprotein, phosphoprotein, matrix protein, hemagglutinin neuramindase protein, fusion protein and large protein were obtained by reverse transcription and PCR methods based on our previous work of sequencing full length genome of sendai virus BB1 strain （DQ219803 in GenBank）. Sequencing showed the six genes were completely identical to that we reported. In order to supply the function necessary for rescuing and packaging of sendai virus vector in trans,the N, P, M, F, HN and L genes were separately cloned into an adenoviral shuttle expression vector pDC316 resulting in six recombinant adenoviral plasimds. Six replicating defective recombinant adenoviruses Ad5-N, Ad5-P, Ad5-M, AdS-F, AdS- HN and Ad5-L were obtained by separately cotransfection of pDC316 carrying N, P, M, F, HN and L genes with the adenoviral genomic plasmid pBHGlox△E1,3Cre into HEK293cells. Restrictive enzymatic results indicated that the six recombinant plasmids were correctly constructed. PCR results showed the recombinant adenoviruses contained the respective SeV genes . Western blotting as well as immunofluorescence assay indicated the expression of the corresponding proteins of sendai virus. These work laid the basis for the construction of the full length genome plasmid of sendai virus BBlstrain and the setup of SeV virus vector system based on SeV BB1 strain.

关 键 词：副粘病毒科 仙台病毒 BB1株 腺病毒载体 

分 类 号：R373.9[医药卫生—病原生物学]