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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。
OBJECTIVE: To clone and prokaryote the newly discovered CsClA2 gene of Clonorchis sinensis to determine if it is an excretory-secretory protein (ESP). Methods CsPLA2-like gene was identified from the full-length cDNA plasmid library of Clonorchis sinensis using a variety of bioinformatics analysis software to analyze the biological function of the expressed protein. CsPLA2 was cloned into prokaryotic expression plasmid pET28a (+), And expressed in Escherichia coli BL21 / DE3. The expressed product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot was used to determine whether CsPLA2 was ESP. Immunofluorescence Methods The tissue localization of CsPLA2 in adults was observed. Results The gene encoded 307 amino acids and contained the signal peptide sequence with PLA2 domain and activation site. The homology was 45% and the similarity was 52%. The results of PCR, double enzyme digestion and DNA sequencing showed that the recombinant plasmid pET28a-CsPLA2 was constructed successfully. SDS-PAGE showed that the target gene was highly expressed in Escherichia coli BL21 / DE3 and the expressed product was inclusion body. Western blot results showed that CsPLA2 is one of the ESP components of Clonorchis sinensis. Immunohistochemical localization of the parasite revealed that CsPLA2 was located in the abdominal sucker, intestine and testis of the parasites. Conclusion A new gene of CsPLA2 was found. The expressed protein was located in the abdominal sucker, intestine and testis of the parasites and was one of the components of adult Eschococcus sinensis adults. This gene is highly expressed in prokaryotic expression systems.