阴道毛滴虫肌动蛋白cDNA克隆与表达

目的克隆阴道毛滴虫肌动蛋白基因片段,表达及纯化肌动蛋白重组蛋白,免疫豚鼠获得抗血清,为进一步研究肌动蛋白在阴道毛滴虫体内定位及其在细胞衰老过程中的作用奠定基础。方法应用PCR方法获得阴道毛滴虫肌动蛋白基因部分片段,并克隆入表达载体pET-41a,转化大肠埃希菌(E.coli)BL21,IPTG诱导重组质粒表达,亲和层析纯化表达产物,Brandford法测定重组蛋白含量并免疫豚鼠,用SDS-PAGE和Western-blot进行分析鉴定。结果肌动蛋白基因的PCR产物片段大小500bp;构建了阴道毛滴虫肌动蛋白基因重组表达质粒;表达了肌动蛋白重组蛋白;SDS-PAGE显示分离纯化的肌动蛋白重组蛋白为47000u;Western-blot结果表明所获得抗血清可与肌动蛋白重组蛋白在47000u及阴道毛滴虫总蛋白在41000u处特异性反应。结论重组肌动蛋白获得高效表达,其免疫豚鼠获得的抗血清可与阴道毛滴虫肌动蛋白重组蛋白反应,也可识别阴道毛滴虫虫体肌动蛋白,该抗血清可用于进一步的研究。

Cloning and Expression of An Actin cDNA Fragment from Trichomonas vaginalis

Objective To clone and express an actin cDNA fragment, and to prepare the antiserum for functional analysis of actin in the formation and maintenance of cystoskeleton in Trichomonas vaginalis. Method A fragment of actin gene was amplified by a pair of primer designed according to an actin cDNA sequence from GenBank. The PCR products were cloned to pGEM-T-Easy vector, and subcloned to the expression vector pET-41. E.coli BL21 was transformed by the recombinant plasmid. The expression of recombinant protein was induced by IPTG.The recombinant protein was purified with GSTrap affinity columns. A guinea pig was injected with the recombinant protein for the preparation of antiserum. SDS-PAGE and Western-blot analysis were performed to detect the recombinant protein. Result A cDNA fragment of 500 bp was cloned, and the recombinant expression plasmid was constructed. Recombinant protein with a molaecular mass of 47000 u was expressed and purified. The antiserum against the recombinant protein was also prepared. Conclusion The recombinant actin protein was successfully expressed. Antiserum for this protein may be used to analyze the function of actin in the formation and maintenance the cystoskeleton in T.vaginalis.