重组乳酸杆菌中A组轮状病毒VP7基因表达  

Expression of group A rotavirus VP7 gene in recombined Lactobacillus

作　　者：吴景华[1] 王海欣[1] 赵杰[1] 刘丽娜[1] 

机构地区：[1]华北煤炭医学院附属医院检验科,河北唐山063000

出　　处：《中国公共卫生》2009年第10期1266-1267,共2页Chinese Journal of Public Health

基　　金：唐山科技局课题(08130227C)

摘　　要：目的扩增A组轮状病毒VP7基因,构建pEDM27/5-VP7重组质粒,转化乳酸杆菌并分析VP7蛋白的表达及活性。方法通过逆转录聚合酶链反应(RT-PCR)获得目的基因VP7,纯化后与pEDM27/5载体进行连接,转化乳酸杆菌,乳糖进行诱导表达,分别于4,8,12 h后,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹技术(Western blot)对产物进行分析测定。结果A组轮状病毒经RT-PCR扩增后,1%琼脂糖电泳检测可见一条约1.0 kb的特异性条带,与预期目的基因(VP7基因)大小相符;SDS-PAGE和Western-blot杂交分析发现,经乳糖诱导4,8,12 h后分别可见一条分子量约为28 kD蛋白带表达,经扫描分析,诱导后表达蛋白分别约占菌体总蛋白的2.30%,5.12%,5.38%,且随着时间变化而持续表达增加,并能与抗A组轮状病毒VP7蛋白特异结合。结论重组乳酸杆菌持续表达免疫活性A组轮状病毒VP7蛋白,为进一步研究乳酸杆菌作为轮状病毒基因工程疫苗的表达载体提供了基础依据。Objective To construct vector pEDM27/5-VP7 by RT-PCR and transform it into Lacotobacillus for the construction of genetic engineering vaccine against rotavirus. Methods The VP7 gene fragment was isolated and recoveried by RT-PCR, and transformed into pEDM27/5. The gene was transformed into Lactobacillus through extraction from the plasmid. After lactose induction, the vp7 was detected with SDS-PAGE and Western-blot at 4,8 and 12 hours. Results The VP7 gene was transformed into pEDM27/5 successfully, and the susceptibity bacteria was recombined by pEDM27/5-vp7. After lactose induction at 4,8,12 hours, the protein（28kD） was detected with SDS-PAGE and Western-blot. The expressed protein was 2. 30% ,5. 12% ,5. 38% of total protein of the bacteria,respectively by scanning,and the expression increased with the increment of the time. The protein could combined it specially combined with anti-vp7 protein. Conclusion The recombinant Lactobacillus expresses immunocompetent vp7 protein constantiy, and could be used for further development of genetic engineering vaccine against rotavirus.

关 键 词：A组轮状病毒 逆转录聚合酶链反应(RT-PCR) 乳酸杆菌 基因转化 

分 类 号：R373.2[医药卫生—病原生物学]