华支睾吸虫19kDa分泌型蛋白(CsSP19)基因的克隆、表达与免疫学功能研究

目的对一个新发现的华支睾吸虫分泌型蛋白的未知基因,进行克隆表达和重组产物的免疫学鉴定,确定其免疫学功能。方法利用生物信息学方法从华支睾吸虫成虫全长cDNA质粒文库中发现一个编码分泌型蛋白的新基因（克隆号Cs004f03）,将去除信号肽序列的编码序列克隆到原核表达质粒pET-28a（＋）中,IPTG诱导表达,产物经亲和层析纯化,并制备大鼠免疫血清,Western-blot方法分析其抗原性和分泌性。结果CsSP19开放阅读框（ORF）有564bp,编码188个氨基酸,理论分子量为21.2kDa,成熟肽的理论分子量为19kDa。该基因的成熟肽编码区在大肠杆菌中可获得高表达,重组蛋白能被华支睾吸虫病人血清所识别,同时,重组蛋白免疫血清也能识别华支睾吸虫分泌排泄抗原中分子量约19kDa的蛋白条带。结论CsSP19蛋白是华支睾吸虫分泌排泄抗原中一个有潜在诊断价值的分泌蛋白。

Cloning,expression and immunological characteristics of the 19 kDa secretory protein from Clonorchis sinensis

A cDNA clone encoding a novel secretory protein was isolated from adult Clonorchis sinensis full length cDNA plasmid library.The sequence encoding the mature peptide was cloned into prokaryotic expression vector pET-28a( )and expressed in E.coli BL21/DE3 after induction with IPTG.Meanwhile,the expressed product after purification with affinity chromatography was used as antigen to prepare rat immune sera.The antigenicity and the secretory property of this protein(CsSP19)were analyzed by Western blotting.It was demonstrated that the open reading frame(ORF)was constituted by 564 bps of nucleotides,encoding 188 amino acids with conceptual molecular weight of 21.2 kDa and the theoretical molecular weight of the mature peptide was 19 kDa.The enoding sequence of the mature peptide could be highly expressed in E.coli.The recombinant protein could be recognized by sera of patients with C.sinensis infection.In addition,sera from rats immunized with the recombinant protein could recognize the secretory protein with molecular weight of 19 kDa in the excretory-secretory antigens of C.sinensis.These results suggest that CsSP19 is a component of excretory-secretory antigens of C.sinensis and may be a potential diagnositic antigen.