日本血吸虫酪氨酸羟化酶基因的克隆、真核表达及鉴定

Cloning of Schistosoma japonicum tyrosine hydroxylase and its eukaryotic expression and identification

目的扩增日本血吸虫的酪氨酸羟化酶(Schistosoma japonicumTyrosine Hydroxylase,Sj TH)编码基因,构建pcDNA3.1(+)-SjTH真核表达载体,并检测其在COS-7细胞中的表达情况。方法以日本血吸虫成虫cDNA为模板,RACE PCR扩增Sj TH编码基因,并与pGEM-T连接进行亚克隆,双酶切后回收目的基因,并与真核表达载体pcDNA3.1(+)连接,PCR和双酶切初步鉴定后测序,纯化无内毒素重组质粒pcDNA3.1(+)-Sj TH,转染入COS-7细胞,G418筛选阳性克隆,RT-PCR和Western blot鉴定重组Sj TH蛋白的表达。结果 RACE PCR扩增出SjTH编码基因,大小约1392bp,经双酶切鉴定、测序及Blast分析鉴定重组真核质粒构建成功。脂质体介导无内毒重组真核质粒pcDNA3.1(+)-SjTH转染入COS-7细胞,G418筛选出阳性克隆,RT-PCR证实阳性单克隆细胞带有SjTH编码基因,Western blot鉴定单克隆细胞表达重组SjTH蛋白,大小约54kD。结论真核表达载体pcDNA3.1(+)-SjTH构建成功,G418筛选出阳性克隆,真核表达重组SjTH蛋白,为后续研究SjTH蛋白功能奠定基础。

The purpose of this study is to clone SjTH gene and construct the pcDNA3.1（＋）-SjTH plasmid to detect its expression in COS-7 cells after being subcloned into pGEM-T vectors,in order to provide the foundation for further studies on its biological activities in the life Cycle of Schistosoma japonicum.Extracting the total RNA from Schistosoma japonicum adult worms and reversing to transcript it to cDNA,the gene encoding protein SjTH was amplified from the cDNA by RACE PCR and then cloned into the plasmid pGEM-T vector.After digesting from recombinant,the SjTH fragments pGEM-T-SjTH was connected with eukaryotic expression vector pcDNA3.1（＋）.The recombinant plasmid was identified by PCR and sequenced.After the endo free pcDNA3.1（＋）-SjTH plasmids were transfected into COS-7 cells,the expression of recombinant protein SjTH could be detected by RT-PCR and Western blot by screening with G418.The gene encoding protein SjTH was amplified by RACE PCR and identified to be about 1 392bp.The combinant pcDNA3.1（＋）-SjTH was confirmed successfully by digesting and sequencing.Endo free plasmid pcDNA3.1（＋）-SjTH were transfected into COS-7 by liposome.The positive clones was screened by G418 and identified by RT-PCR.The combinant SjTH protein was expressed in positive cloned cell with pcDNA3.1（＋）-SjTH and identified to be about 54kDa by Western blotting.The plasmid pcDNA3.1（＋）-SjTH was constructed successfully and could express the recombinant SjTH protein after transfecting into COS-7 cells.The studies lay a foundation for function research of SjTH subsequently.