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克隆日本血吸虫原肌球蛋白编码基因 ,并在大肠杆菌中表达。方法 抽提日本血吸虫 (大陆株 )成虫总RNA ,经逆转录—聚合酶链反应 (RT PCR)获得编码日本血吸虫原肌球蛋白的cDNA片段 ,该片段与全序列比较 ,缺氨基端 1 4个氨基酸。该PCR产物克隆入T载体并对插入片段进行序列测定后 ,亚克隆入表达载体pbV2 2 0 ,经琼脂糖凝胶电泳、限制性酶切反应和PCR鉴定后 ,选择克隆用于温控表达。结果 RT PCR产物克隆入T载体 ,序列测定表明 ,在核苷酸水平和推断和氨基酸水平 ,与曼氏血吸虫分别有96 5%和 98 1的同源性。目的DNA片段亚克隆入原核表达载体pBV2 2 0 ,在大肠杆菌DH5α中诱导表达。经SDS PAGE和Westernblot分析表明 ,该非融合表达产物相对分子质量约为 3 2 0 0 0 ( 3 2kDa) ,日本血吸虫 (大陆株 )成虫天然原肌球蛋白免疫兔血清能特异识别该重组蛋白。结论 编码日本血吸虫原肌球蛋白cDNA克隆及起其在细菌中的表达获得成功
The S. japonicum tropomyosin encoding gene was cloned and expressed in E. coli. Methods The total RNA of adult Schistosoma japonicum (Chinese strain) was extracted, and the cDNA fragment coding for myosin of Schistosoma japonicum was obtained by reverse transcriptase - polymerase chain reaction (RT PCR). Compared with the full - length sequence, Amino acids. The PCR product was cloned into T vector and sequenced. The PCR product was subcloned into expression vector pbV220. After cloning by agarose gel electrophoresis, restriction enzyme digestion and PCR, clones were selected for temperature-dependent expression. Results The RT PCR product was cloned into T vector. Sequence analysis showed 96.5% and 981 homologies with Schistosoma mansoni at nucleotide level and deduced amino acid level respectively. The target DNA fragment was subcloned into the prokaryotic expression vector pBV220 and induced in E. coli DH5α. SDS PAGE and Western blot analysis showed that the relative molecular mass of the non-fusion product was about 32,000 (32 kDa). The purified product of the native tropomyosin immunized rabbit serum of Schistosoma japonicum (Chinese strain) could specifically recognize the recombinant protein. Conclusion The cDNA encoding Schistosoma japonicum tropomyosin cDNA was successfully cloned and its expression in bacteria was successful