大片吸虫α-微管蛋白基因的克隆表达及免疫反应性的研究

Cloning,expression and immune-reactivity of α-tubulin gene of Fasciola gigantica

为鉴定大片吸虫α-微管蛋白基因(FgAT),并分析其作为诊断抗原的可行性,根据肝片吸虫的基因组及转录组数据设计α-微管蛋白基因的扩增引物,以大片吸虫总RN A为模板,通过RT-PCR获得大片吸虫的FgAT基因;对其进行生物信息学分析及鉴定,然后构建pGEX-6P-1(+)-FgAT重组表达载体,在大肠杆菌BL21(DE3)中进行诱导表达,再对纯化的目的蛋白进行SDS-PAGE和Western-blot分析。结果表明,大片吸虫FgAT基因的大小为1 356 bp,编码452个氨基酸残基。生物信息学分析表明,FgAT二级结构预测以无规卷曲为主,抗原表位在整个序列中均有存在。优化后的α-微管蛋白为可溶性表达,Western-blot分析结果表明其具有良好的免疫反应性。本研究首次克隆并鉴定了大片吸虫α-微管蛋白基因,表达的重组蛋白具有良好的免疫反应性,该蛋白是潜在的大片吸虫诊断靶标。

To identify α-tubulin gene of Fasciola gigantica（FgAT） and analyze its feasibility as a diagnostic antigen,the specific primers were designed according to the genome and transcriptome data of F.hepatica,and the complete ORF was amplified from total RNA by RT-PCR.The fragment including the FgAT complete ORF was cloned into the p GEX-6P-1 expression vector.The α-tubulin fusion protein was expressed in Escherichia coli and immune-reactivity was tested by Western-blot.The results showed thatα-tubulin gene sequence contained an ORF of 1 356 bp,encoding a protein of 452 amino acids.Bioinformatics analysis showed that the secondary structure of Fg AT was mainly regulated by random coil,and the antigen epitopes were present in the whole sequence.Western-blot using rabbit antibodies against F.gigantica excretory and secretory products（FgESPs） indicated good immune-reactivity.This study cloned and expressed the fragment α-tubulin for the first time,and the recombinant protein has immune-reactivity,indicating that F.gigantica α-tubulin may represent a candidate antigen for the diagnosis ofF.gigantica infection.