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

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-T Easy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43 000.结论 成功进行了BmGAPDH编码基因的克降和真核表达.Cloning,weukaryotic expremion of the gene encoding glyceraidehydes-3-phosphate dehydrogenase from periodic Brugia malayi XIE Dong-fimg,FANG Zheng,HUANG Wei-qun,SHEN Qin,TONG Hai-yan,XUBang-sheng.%Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase(GAPDH)from periodic Brugia molayi(Bm).Methods Total RNA was extraeted from periodic Brugic malayi.The BmGAPDH gene was amplified by RT-PCR.The PCR product was cloned and then subeloned 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 highiy expressed in transfected COS-7 cell.The deduced amino acid sequence was identical with that of BmGAPDH.The recombinant pnotein wag about Nr 43 000.Conclusion The recombinant plasmid peDNA3.1(+)-BmGAPDH has been constructed and the protein has been expressed correctly.