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当前位置: 首页 > 文章 > 乳房链球菌GAPC基因的克隆及原核表达 动物医学进展 2008,29 (6) 62-65
Position: Home > Articles > Cloning and Prokaryotic Expression of Gene Encoding GAPC of Streptococcus uberis Progress in Veterinary Medicine 2008,29 (6) 62-65

乳房链球菌GAPC基因的克隆及原核表达

作  者：
褚明亮;陈创夫;刘君;曹旭东
单  位：
新疆地方与民族高发病省部共建教育部重点实验室;新疆石河子大学医学院;新疆石河子大学动物科技学院
关键词：
乳房链球菌;表面脱氢酶GAPC;原核表达
摘  要：
为了重组表达乳房链球菌表面脱氢酶GAPC基因,为免疫学研究提供目标蛋白,用PCR方法从石河子分离株的基因组DNA中扩增出GAPC基因,用T/A克隆法将其插入PBST载体,并构建原核表达载体pET-32a(+)-GAPC。用BL21(DE3)/pET系统表达Trix-GAPC融合蛋白,SDS-PAGE和Western blot分析鉴定表达产物。结果表明,PCR扩增产物经测序,证实与GenBank中乳房链球菌(AF421900.1)GAPC的基因序列同源性为99%。SDS-PAGE显示,经IPTG诱导后BL21(DE3)/pET-32a(+)-GAP总蛋白中出现一条分子质量为55ku的新蛋白带。Western blot分析显示,GAPC蛋白可与乳房链球菌多克隆抗血清发生特异性反应。该研究已成功表达了GAPC,为GAPC在细菌致病中作用的研究以及相关疫苗的制备奠定了基础。
译  名：
Cloning and Prokaryotic Expression of Gene Encoding GAPC of Streptococcus uberis
作  者：
CHU Ming-liang1,2,CHEN Chuang-fu1,3,LIU Jun3,CAO Xu-dong4 (1.Ministry of Education Key Laboratory of Xinjiang Endemic and Ethnic Disease,Shihezi,Xinjiang,832003,China;2.College of Life Sciences,Shihezi University,Shihezi,Xinjiang,832003,China;3.College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832003,China4.Medical School,Shihezi University,Shihezi,Xinjiang,832003,China)
关键词：
Streptococcus uberis;glyceraldehyde-3-phosphate dehydrogenase;prokaryotic expression
摘  要：
To express the gene encoding the glyceraldehyde-3-phosphate dehydrogenase(GAPC) of Streptococcus uberis and provide target protein for further immunological study,the GAPC gene from the genomic DNA of Streptococcus uberis strain isolated from cow in Shihezi was amplified by PCR and inserted into PBST vector by T/A cloning.The recombinant plasmid was digested with restriction endonuclease BamHⅠ and SalⅡ,and the obtained gene fragment was cloned into pET-32a(+) vector.The constructed recombinant plasmid pET-32a(+)-GAPC was transformed into BL21(DE3)/pET system for expression of Trix-GAPC fusion protein.The expressed product was identified by SDS-PAGE and Western blot. Results showed that the homology of amplified GAPC gene sequence to that of GAPC of Streptococcus uberis(AF421900.1) reported in GenBank was 99%.SDS-PAGE showed a protein band with relative molecular weight of 55 ku.The activity of recombinant protein was analyzed by Western blot. In conclusion Streptococcus uberis GAPC fusion protein was successful expressed.It laid a foundation for study on role of GAPC in pathogene of bacteria and development of relevant vaccine.

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