阴道毛滴虫病毒衣壳蛋白基因cap_(2037)的克隆与表达

Cloning and expression of the capsid protein gene cap_(2037) of Trichomonas vaginalis

目的对阴道毛滴虫病毒衣壳蛋白基因的克隆与原核表达。方法提取阴道毛滴虫总核酸为模板,根据所克隆的阴道毛滴虫病毒部分序列和GenBank中发表的阴道毛滴虫病毒TvV-T1序列设计一对引物,经RT-PCR得到与预计大小一致的PCR特异性产物,将其克隆到pMD-18T载体后测序,并与GenBank中核苷酸序列进行同源性搜索与分析,再将其克隆至表达载体pET28a。并以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果目的基因片段长度为2037bp,与阴道毛滴虫病毒T1株(U08999)的同源性最高为82.9%,构建原核表达载体pET-Cap2037;IPTG诱导后,SDS-PAGE显示表达产物的大小约75kDa。结论成功克隆出阴道毛滴虫病毒衣壳蛋白基因序列,与TVV-T1株序列有82.9%的同源性,经IPTG诱导,SDS-PAGE分析表明,75kDa蛋白基因在大肠杆菌BL21(DE3)中得到高效表达。

To clone and express the capsid protein gene cap2037 of Trichomonas vaginalis, a pair of primers was designed according to the cloned partial gene of T. vaginalis virus and the published sequence of the T1 strain of T. vaginalis virus （U08999 in GenBank）. The total nucleic acid extracted from the trophozoites of this parasitte was used as template for the amplification of the viral capsid genome by using the RT-PCR technique. Then the amplified product was cloned to vector pMD18- T, sequenced and compared with the sequence available in GenBank. Positive clones were subcloned to the expression plasmid pET28a（＋） ,and the recombinant plasmid was transformed to E. coli BL21 （DE3） cells. The recombinant clones after characterization with PCR and digestion by restriction endonucleases were inducted with IPTG to express the target protein, and the expressed protein was characterized by SDS-PAGE. It was demonstrated that the length of the target gene fragment was 2037 bps. This gene fragment shared 82.9 % of sequence identity with the T1 strain of T. vaginalis virus . High expression of the 75 kDa target protein could be obtained in E. coli BL21（DE3） cells.