旋毛虫果糖-1,6-二磷酸酶基因的克隆表达及生物学特性分析

Clone,expression and biological characteristics of Trichinella spiralis fructose-1,6-bisphosphatase

根据Gen Bank公布的旋毛虫果糖-1,6-二磷酸酶(FBP)基因序列,设计1对特异性引物,以旋毛虫总RNA为模板,用RT-PCR技术扩增出1条约为1 kb的DNA片段。测序结果表明该片段包含FBP的完整ORF,长1 026 bp,编码341个氨基酸。BLAST分析发现其核酸和氨基酸序列与已知旋毛虫FBP的基因和氨基酸序列的相似性分别为99%和100%。将该ORF亚克隆到p ET-32a(+)载体中,酶切鉴定正确后转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析,结果表明该融合蛋白大小为55.4 ku,重组蛋白主要以包涵体的形式表达。Western blot结果显示该重组蛋白能被旋毛虫感染小鼠血清识别。对纯化、复性的重组蛋白进行了酶活性测定,结果发现该重组酶的最适p H为7.0,最适温度为37℃。

To investigate the biological characteristics of fructose-1, 6- bisphosphatase （FBP）, cDNA from Trichinella spiralis was cloned and expressed. To amplify the target gene, a pair of gene specific primers was designed based on the FBP ORF, and RT-PCR was performed. The PCR product was cloned into pMD19-T vector. Sequence analysis showed that the FBP gene contains an ORF of 1 026 bp and encodes 341 amino acids. Results of BLAST proved that the gene sequence was highly homology with that available on the GenBank. The FBP gene was further subcloned into pET-32a vector and transferred into Escherichia coli BL21 （DE3）. To induce the expression, IPTG was added to the bacterial cultures. SDS-PAGE analysis showed that the recombinant protein TsFBP with the molecular weight of 55.4ku was expressed in the insoluble inclusion body. Western blot analysis indicated that the recombinant TsFBP can be recognized by serum from the T. spiralis infected mice. Enzyme catalytic experiment indicated that the optimum pH was 7.0 and the optimum temperature was 37℃ for the recombinant protein.