日本血吸虫HGPRT编码基因的克隆与表达

Cloning and expression of the gene encoding hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum

目的克隆和表达日本血吸虫大陆株次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)编码基因。方法依据GenBank日本血吸虫HGPRT开放阅读框(ORF)设计一对引物,上游和下游引物分别引入BamH和Sal酶切位点。以日本血吸虫大陆株(安徽株,简称Sjc-A)成虫总RNA为模板,反转录PCR(RT-PCR)扩增日本血吸虫大陆株HGPRT(SjcHGPRT)全长编码基因。经双酶切纯化的PCR产物与同样双酶切纯化的pET28a质粒DNA片段用T4DNA连接酶连接,构建重组质粒pET28a-SjcHGPRT,转化感受态E.coli BL21,并大量扩增。重组质粒DNA经限制性内切酶双酶切、PCR、琼脂糖凝胶电泳和核苷酸序列测定进行鉴定。pET28a-SjcHGPRT/E.coli BL21工程菌用IPTG诱导表达,重组蛋白用SDS-PAGE和Western blot分析。结果SjcHGPRT编码基因RT-PCR产物约700bp,构建的pET28a-SjcHGPRT重组质粒DNA经限制性内切酶双酶切和PCR扩增产物于琼脂糖凝胶电泳均观察到相同大小的基因片段,根据核苷酸序列测序结果推导的氨基酸序列与报道的日本血吸虫大陆株(湖南株,简称Sjc-H)及曼氏血吸虫HGPRT分别有99%和83%的同源性。获得的重组蛋白(reSjcHGPRT)经SDS-PAGE和Western blot分析,分子量约30kDa,并能被抗His-G-HRP抗体、日本血吸虫感染小鼠血清和日本血吸虫病人血清识别。结论日本血吸虫大陆株(安徽株)HGPRT表达获得成功,并获得了纯化重组蛋白,为开展该分子功能和免疫原性研究奠定了基础。

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chi- nese-strain hypoxanthine-guanine phosphoribosyltransferase （HGPRT） and its expression in Escherichia coli. Methods A couple of primers were designed with the BamHⅠ restriction endonuclease site introduced in forward primer and Sal Ⅰ in reverse primer. Total RNA was isolated from adult worms of S. japonicum Chinese-strain（Anhui-strain, Sjc-A）and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR）. The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHⅠ and SalⅠ. The target DNA fragments were purified and cloned properly into pET28a. After identification by en-donucleases digestion, PCR and sequencing, the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E. coli BL21 and expressed in the presence of IPTG. Results pET28a- SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S. japonicurn Chinese-strain （Hunan-strain, Sjc-H） and S. rnansoni HGPRT, respectively. The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica. Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein （reSjcHGPRT） is also successfully purified.