中文摘要：

目的克隆并表达刚地弓形虫热休克蛋白70（TgHsp70）编码基因,并对重组TgHsp70（rTgHsp70）的抗原表位进行生物信息学分析。方法收集、纯化RH株弓形虫速殖子,提取总RNA,RT-PCR扩增TgHsp70编码基因,插入原核表达质粒pET30a（＋）中,转化大肠杆菌BL21（DE3）,IPTG诱导表达,表达产物经SDS-PAGE、Western blot进行检测,并采用相关生物信息学软件对重组蛋白结构和表位特征进行分析。结果从弓形虫RH株cDNA中扩增出495bp的TgHsp70部分编码基因,重组表达质粒pET30a（＋）/TgHsp70经PCR和双酶切鉴定构建正确;目的基因在大肠杆菌BL21（DE3）中高效表达,rTgHsp70相对分子质量约18000,其能被兔抗弓形虫血清识别;rTgHsp70存在多个功能位点及潜在的抗原决定簇。结论已在原核表达系统中高效表达了RH株TgHsp70,该重组蛋白有多个抗原表位,具有抗原性,有望作为弓形虫疫苗的候选抗原。

英文摘要：

Objective To clone and express the gene encoding the heat shock protein 70 of Toxoplasma gondii（TgHsp70） and analyze the bioinformatics of the recombinant protein.Methods Toxoplasma gondii tachyzoites of RH strain were collected and purified,from which total RNA was extracted,and the encoding gene of TgHsp70 was amplified by RT-PCR and inserted into prokaryotic expression vector pET30a（＋）.The constructed recombinant plasmid pET30a（＋）/TgHsp70 was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,of which the structure and epitope characteristics were analyzed by the relevant bioinformatics software.Results Partial gene encoding TgHsp70,at a length of 495 bp,was amplified from the cDNA of T.gondii RH strain,and recombinant plasmid pET30a（＋）/TgHsp70 was constructed correctly as proved by PCR and restriction analysis.The target gene was highly expressed in E.coli BL21（DE3）,and the expressed recombinant TgHsp70,with a relative molecular mass of about 18 000,was recognized by rabbit antisera against T.gondii.The rTgHsp70 possibly had multiple domains and potential antigenic determinants.Conclusions The TgHsp70 of RH strain was successfully expressed in prokaryotic expression system,which had multiple antigenic determinants and showed antigenicity,and might be used as a candidate antigen of T.gondii vaccine.