周期型马来丝虫3-磷酸甘油醛脱氢酶编码基因的克隆与真核表达  

Cloning, weukaryotic expression of the gene encoding glyceraldehydes-3-phosphate dehydrogenase from periodic Brugia malayi

作　　者：谢东方[1] 方政[1] 黄为群[1] 沈勤[1] 童海燕[1] 徐邦生[1] 

机构地区：[1]南通大学医学院寄生虫学教研室,226001

出　　处：《中国地方病学杂志》2008年第6期609-612,共4页Chinese Jouranl of Endemiology

基　　金：江苏省社会发展科技计划项目（BS2006522）

摘　　要：目的克隆和表达周期型马来丝虫3-磷酸甘油醛脱氢酶（BmGAPDH）的编码基因。方法依据公布的BmGAPDH基因序列设计引物，以周期型马来丝虫总RNA为模板，反转录PCR（RT-PCR）扩增目的编码基因。PCR产物经TA克隆后，克隆至载体pGEM-TEasy中，经PCR和双酶切鉴定后，亚克隆至真核表达质粒pcDNA3．1（＋），构建真核表达载体pcDNA3．1（＋）／BmGAPDH，转染COS-7细胞后进行RT-PCR验证。用十二烷基磺酸钠-聚丙烯酰胺电泳（SDS-PAGE）对获得重组蛋白（rBmGAPDH）进行分析和鉴定。结果转染的COS-7细胞高水平表达BmGAPDH的mRNA，根据克隆的目的基因序列推导的氨基酸序列与GenBank登录的结果一致。SDS-PAGE分析显示重组蛋白rBmGAPDH相对分子质量（Mr）约为43000。结论成功进行了BmGAPDH编码基因的克隆和真核表达。Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase （GAPDH） from periodic Brugia malayi （Bm）. Methods Total RNA was extracted from periodic Brugia malayi. The BmGAPDH gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pcDNA3.1（＋） vector. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into COS-7 cell subsequently. The expressed protein was identified by SDS-PAGE. Results BmGAPDH mRNA was highly expressed in transfected COS-7 cell. The deduced amino acid sequence was identical with that of BrnGAPDH. The recombinant protein was about Mr 43 000. Conclusion The recombinant plasmid pcDNA3.1 （＋）-BmGAPDH has been constructed and the protein has been expressed correctly.

关 键 词：周期型马来丝虫 3-磷酸甘油醛脱氢酶 真核表达载体 COS-7细胞 

分 类 号：R686[医药卫生—骨科学]