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目的 扩增日本血吸虫 1 4 3 3蛋白epsilon亚型编码基因并构建其表达载体 ,以研究其在血吸虫信号转导以及免疫预防和诊断方面的作用。方法 以日本血吸虫成虫总RNA为模板逆转录合成cDNA链 ,设计合成引物 ,用PCR法扩增 1 4 3 3蛋白epsilon亚型基因编码序列 ,将其克隆入pGEM T载体 ,双酶切和以质粒为模板的PCR鉴定后 ,将基因片段亚克隆入表达载体pBK CMV ,转染大肠杆菌BL2 1 ,经双酶切后鉴定。结果 RT PCR扩增出一条约 753bp的特异性条带 ,TA克隆重组质粒的双酶切和以质粒为模板的PCR均获得了一条与RT PCR扩增出大小相同条带 ,pBK Sj1 4 3 3表达载体经双酶切后也同样获得了一条约 753bp的特异性条带。 结论 在体外成功的扩增了日本血吸虫 1 4 3 3蛋白epsilon亚型编码基因并构建了 pBK Sj1 4 3 3表达载体 ,为进一步的免疫预防和免疫诊断研究提供了条件。
Objective To amplify the gene encoding epsilon subfamily of 1443 protein of Schistosoma japonicum and construct its expression vector to study its role in the signal transduction of schistosomes and in the prevention and diagnosis of immune diseases. Methods cDNA was synthesized by reverse transcription of the total RNA of Schistosoma japonicum as a template. The primers were designed and synthesized. The coding sequence of the epsilon subunit of 1 433 protein was amplified by PCR and cloned into pGEM T vector. After PCR identification of the template, the gene fragment was subcloned into the expression vector pBK CMV and transfected into E. coli BL21, and identified by double enzyme digestion. Results A specific band of about 753bp was amplified by RT-PCR. A double-digested cDNA fragment of TA clone and plasmid-based PCR were obtained. After double digestion, the expression vector also obtained a specific band of about 753bp. Conclusion The gene encoding epsilon subunit of 1 433 protein of Schistosoma japonicum was successfully amplified in vitro and the pBK Sj1 4 3 3 expression vector was constructed, which provided the conditions for further immunoprevention and immunodiagnosis.