弓形虫RH株ROP38基因的原核表达及其反应原性研究

Prokaryotic expression and reactinogenicity characterization of ROP38 gene of Toxoplasma gondii RH strain

为了研究弓形虫ROP38(TgROP38)蛋白的性质和反应原性,以弓形虫RH株基因组DNA为模板,应用聚合酶链反应(PCR)扩增TgROP38基因。测序后,将核苷酸序列翻译成氨基酸序列并利用生物信息学软件进行分析。将测序正确的片段连接至pET-30a(+)表达载体后,进行原核表达,并用Western-blot检测其反应原性。结果表明,成功扩增出大小约为1 600bp的TgROP38基因。将其翻译成氨基酸序列后,经生物信息学软件分析得出TgROP38蛋白是跨膜蛋白,含有多个亲水区域和B细胞表位。SDS-PAGE分析和Western-blot分析表明,TgROP38蛋白的大小约为66ku,且具有较好的反应原性。结果表明,TgROP38蛋白可作为抗弓形虫病疫苗候选分子研制弓形虫病亚单位疫苗及表位疫苗。

In order to express Toxoplasma gondii ROP38 protein（TgROP38） in vitro and study its re- actinogenicity,TgROP38 gene was amplified by PCR from the genomic DNA of T. gondii RH strain. After sequencing,the nucleotide sequence of TgROP38 gene was putatively translated into amino acids and ana- lyzed by bioinformatics software. The PCR product was ligated into the prokaryotic expression vector pET- 30a（-t-） and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） strain. The recom- binant protein TgROP38 was expressed by inducing with IPTG and its reactinogenicity was analyzed by Western-blot. In result,TgROP38 gene was approximately 1 600 bp in length. SDS-PAGE analysis showed that the recombinant protein was expressed in E. coli with 66 ku in molecular mass,and Western-blot anal- ysis indicated that TgROP38 protein possessed good reactinogenicity. TgROP38 protein was predicted to contain multiple transmembrane and hydrophilic regions and several B cell epitopes,indicating that it could be used as a new vaccine candidate antigen against toxoplasmosis. The results showed that TgROP38 protein was a potential protein for development of molecular vaccines against T. gondii infection.