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目的通过筛选亚洲牛带绦虫(Taenia saginata asiatica)成虫cDNA质粒文库,识别半胱氨酸分泌蛋白(Ta CRISP),并进行克隆表达,为进一步研究其功能提供基础依据。方法用生物信息学方法从亚洲牛带绦虫成虫全长cDNA质粒文库中识别TaCRISP的全长编码基因并预测编码蛋白质的各种结构与功能;将其编码区序列克隆到原核表达载体pET-30a(+)上,测序鉴定重组质粒,之后进行诱导表达,表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。结果该基因全长1090bp,编码239个氨基酸,1aa-16aa位为分泌信号肽,理论分子量为26973.8,等电点为5.96。生物信息分析揭示,Ta CRISP与中殖孔绦虫CRISP一致性为38%,相似性为54%;所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符。SDS-PAGE结果表明,目的基因在大肠埃希菌BL-21/DE3中表达成功。结论发现亚洲牛带绦虫Ta CRISP基因,成功构建重组原核表达质粒并表达出融合蛋白。
OBJECTIVE: To screen TaqMan cDNA of Taenia saginata asiatica and identify Ta CRISP and clone it for further study of its function. Methods The bioinformatics method was used to identify the full-length TaCRISP encoding gene from the full-length cDNA plasmid library of adult Taenia saginata asiatica and to predict the structure and function of the protein. The coding region of TaCRISP was cloned into prokaryotic expression vector pET-30a +). The recombinant plasmids were identified by sequencing and then induced by SDS-PAGE. SDS-PAGE was used to identify the expression products. Results The full length cDNA of this gene was 1090bp, encoding 239 amino acids. The gene laa-16aa was a secretory signal peptide with a theoretical molecular weight of 26973.8 and an isoelectric point of 5.96. Bioinformatics analysis revealed that CRISP of Ta CRISP was identical to that of Taenia solium and the similarity was 54%. The constructed recombinant was identified by PCR, double enzyme digestion and sequencing. SDS-PAGE results showed that the target gene was successfully expressed in Escherichia coli BL-21 / DE3. Conclusion Ta CRISP gene of Taenia saginata was found in Asia, and the recombinant prokaryotic expression plasmid was successfully constructed and the fusion protein was expressed.