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目的:克隆日本血吸虫琥珀酸脱氢酶铁硫蛋白(SjSDISP)全长编码基因,并对所获基因在大肠杆菌中进行表达。方法:根据基因库中SjSDISP对应的EST(BU804141)以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjSDISP基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接成全长cDNA,将其克隆到表达载体pGEX-4T-1上,经琼脂糖凝胶电泳、限制性内切酶消化、PCR和测序鉴定后,选择阳性克隆进行表达及Western印迹分析。结果:获得1071bp全长cDNA,其理论推导为编码278个氨基酸残基的蛋白质。SjSDISP基因在大肠杆菌BL21中获得良好表达。该融合表达产物相对分子质量约为56kD,且能被日本血吸虫成虫抗原免疫血清特异识别。结论:编码日本血吸虫琥珀酸脱氢酶铁硫蛋白全长cDNA克隆及其在大肠杆菌中的表达获得成功。
OBJECTIVE: To clone the full-length coding gene of SjSDISP from Schistosoma japonicum and express the gene in Escherichia coli. Methods: The primers were designed according to the SjSDISP-compatible EST (BU804141) in the gene bank and the adjacent nucleotide sequence of the λgt11 multi-cloning site of adult Schistosoma japonicum cDNA library vector. Using the cDNA library of Schistosoma japonicum as a template, The complete 3 ’end and 5’ end were amplified and sequenced. The full-length cDNA was spliced by electronic software and cloned into the expression vector pGEX-4T-1. After agarose gel electrophoresis and digestion with restriction enzyme, After PCR and sequencing identification, positive clones were selected for expression and Western blot analysis. Results: A 1071 bp full-length cDNA was obtained and deduced theoretically as a protein encoding 278 amino acid residues. The SjSDISP gene was well expressed in E. coli BL21. The relative molecular mass of the fusion expression product is about 56 kD, and can be specifically recognized by the immune serum of adult Schistosoma japonicum antigen. CONCLUSION: The full length cDNA encoding the succinate dehydrogenase of S. japonicum and its expression in E. coli were successfully obtained.