猪传染性胃肠炎病毒HB06株核衣壳蛋白基因的克隆与表达
Cloning and expression of nucleocapsid protein gene of TGEV HB06 strain.
作者信息
Fan Jinghui, Zuo Yuzhu, Zhao Yuelan, Li Tanqing, Zhang Xiaobo
机构信息
1College of Animal Science and Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001 China.
2College of Traditional Chinese Veterinary Medicine, Agricultural University of Hebei, Dingzhou, 073000 China.
出版信息
Front Agric China. 2007;1(3):357-360. doi: 10.1007/s11703-007-0060-5.
The nucleocapsid protein gene of transmissible gastroenteritis virus, 1 149 bp in length, was amplified by RT-PCR from isolated strain HB06 and cloned into pMD18-T. Sequence comparison with other transmissible gastroenteritis virus (TGEV) strains selected from the Gene Bank revealed that the homology of gene complete sequence shares more than 97% in nucleotide. gene was cloned into HI and RI multiple cloning sites of the prokaryotic expression vector pET 20 b, and named pETN. After being induced by isopropyl--D-thiogalactopyranoside (IPTG), the recombinant nucleocapsid protein was expressed. The result of SDS-PAGE and Western-blot showed that the recombinant nucleocapsid protein was 47 kDa and had strong positive reactions with TGEV-specific antibody.
从分离的HB06株中通过RT-PCR扩增出长度为1149bp的传染性胃肠炎病毒核衣壳蛋白基因，并将其克隆到pMD18-T载体中。与从基因库中选取的其他传染性胃肠炎病毒（TGEV）株进行序列比较，结果显示该基因完整序列在核苷酸水平上的同源性超过97%。将该基因克隆到原核表达载体pET 20 b的HindIII和EcoRI多克隆位点中，命名为pETN。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，表达了重组核衣壳蛋白。SDS-PAGE和Western-blot结果表明，重组核衣壳蛋白大小为47kDa，与TGEV特异性抗体有强烈的阳性反应。
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