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目的:构建含有小鼠IL-17A(mIL-17A)基因的重组原核表达载体,获得高效表达mIL-17A的基因工程菌,以及较高产量的mIL-17A蛋白。方法:以PMA活化后小鼠脾脏单个核细胞的总RNA逆转录合成的cDNA为模板,PCR法扩增mIL-17A的编码序列,并分别亚克隆至pMD18-T载体和原核表达载体pGEX-4T-1中,经酶切和DNA测序鉴定后,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,产物经SDS-PAGE及Westernblot鉴定。结果:PCR产物大小及其双酶切鉴定均证明所克隆的基因是mIL-17A,DNA序列测定进一步证实与GenBank报道的序列完全一致。成功构建了重组原核表达载体pGEX-4T-1/mIL-17A,并在大肠杆菌中高效表达出相对分子质量(Mr)约40000的具有可溶性的融合蛋白,且Westernblot证实确为目的蛋白。结论:成功构建了基因重组体pGEX-4T-1/mIL-17A;并制备出可溶性IL-17A-GST融合蛋白。
Objective: To construct a recombinant prokaryotic expression vector containing mouse IL-17A (mIL-17A) gene and obtain the genetically engineered bacteria with high expression of mIL-17A and higher yield of mIL-17A protein. Methods: The cDNA of mIL-17A was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of spleen mononuclear cells of PMA activated cells and cloned into pMD18-T vector and pGEX-4T -1, identified by restriction enzyme and DNA sequencing, transformed into competent E. coli BL21 (DE3), induced by IPTG, the product was identified by SDS-PAGE and Western blot. Results: The size of the PCR product and its double digestion proved that the cloned gene was mIL-17A. The DNA sequencing further confirmed that the sequence was identical with that reported in GenBank. The recombinant prokaryotic expression vector pGEX-4T-1 / mIL-17A was successfully constructed and a soluble fusion protein with a relative molecular mass (Mr) of about 40,000 was efficiently expressed in E. coli, and Western blot confirmed that the target protein was indeed. Conclusion: The recombinant plasmid pGEX-4T-1 / mIL-17A was successfully constructed and soluble IL-17A-GST fusion protein was prepared.