华支睾吸虫磷脂酶A2基因生物学特性的初步研究

目的对新发现的华支睾吸虫磷脂酶A2（CsPLA2）基因进行生克隆、原核表达,确定该蛋白是否为成虫分泌/排泄蛋白（excretory-secretory protein,ESP）。方法利用多种生物信息学分析软件,从华支睾吸虫全长cDNA质粒文库中识别出CsPLA2样基因,分析该基因表达蛋白的生物学功能特征;将CsPLA2克隆入原核表达质粒pET28a（＋）,并在大肠埃希菌BL21/DE3中诱导表达,蛋白表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定;应用Western blot确定CsPLA2是否为ESP;应用免疫荧光方法观察CsPLA2在成虫的组织定位。结果该基因编码307个氨基酸,含有信号肽序列,具有PLA2功能域和活化位点,与谷蛀虫同源蛋白相比,一致性为45%,相似性为52%。PCR、双酶切及DNA测序结果表明pET28a-CsPLA2重组质粒构建成功,SDS-PAGE显示目的基因在大肠埃希菌BL21/DE3中高效表达,表达产物为包涵体。Western blot结果表明CsPLA2是华支睾吸虫的ESP组分之一。虫体免疫组织化学定位显示CsPLA2定位于虫体的腹吸盘、肠支、睾丸处。结论新发现一个CsPLA2基因,其表达的蛋白定位于虫体的腹吸盘、肠支、睾丸处,为华支睾吸虫成虫ESP组分之一。该基因可在原核表达系统中高效表达。

Molecular characterization of a novel phospholipase A2 from Clonorchis sinensis

Objective To clone and express the novel gene phospholipase A2 of Clonorchis sinensis （CsPLA2） for molecular characterization. Methods Using the full length eDNA plasmid clone （No. Cs005f08） as a template, the coding region of phospholipase A2 was amplified by PCR, cloned into the prokaryotic expression vector pET28a （＋）, and then expressed in Escherichia coli BL21 by IPTG induction. The recombinant protein was detected by SDS-PAGE. The excretory-secretory properties of this protein were analyzed by Western blotting. Immunohistoehemical localization of CsPLA2 in the living adults of C. sinensis was done using fluorescence microscopy. Results A eDNA clone encoding a novel phospholipase A2 （307 amino acids） was isolated from a C. sinensis adult eDNA library. Analysis using bioinformaties software showed that the possible sequence shared conserved features of phospholipase A2 and its active site. In a search of GenBank, the signal sequence cleavage site of CsPLA2 was after 36Val. The predicted CsPLA2 amino acid sequence had 45 % identity and 52 % similarity to Triboliurn castaneurn. The prokaryotic expressing vector （pET28a-CsLysoPLA） constructed was successfully indentified by PCR, restriction enzyme digestion, and sequencing. The coding sequence of the gene was highly expressed in E. coll. Western blot analysis indicated that it belonged to excretory-secretory proteins of the adults. Immunohistochemistry suggested that the CsPLA2 was markedly localized in the ventral sucker, intestine, and testis of the adults. Conclusion The novel CsPLA2, a component of excretory-secretory proteins, was localized in the ventral sucker, intestine, and testis of adults. The coding region of CsPLA2 was successfully expressed in E. coll.