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目的:体外构建弓形虫微线体蛋白6(MIC6)突变体并分析其特性。方法:利用基因定点突变的方法,将MIC6蛋白羧基端(MIC6C)的348位色氨酸的碱基突变为缬氨酸的碱基,PCR扩增MIC6C W/V突变体基因片段;构建MIC6C W/V/pGEX-4T-1重组原核表达系统,IPTG诱导表达GST-MIC6C W/V突变体蛋白。以该蛋白为探针蛋白与弓形虫裂解液进行GST沉降实验,SDS-PAGE及Western blot分析。结果:获得了MIC6C W/V突变体基因片段,制备了GST-MIC6C W/V突变体蛋白;MIC6C W/V突变体蛋白的沉降产物中未见蛋白条带,而MIC6C蛋白(未突变蛋白)的产物中有一蛋白条带,且能被抗醛缩酶抗体识别。结论:在体外获得了MIC6突变体;MIC6突变体失去了与醛缩酶作用的特性。
Objective: To construct Toxoplasma gondii MIC6 mutant in vitro and analyze its characteristics. Methods: The mutation of 348 bp tryptophan at the carboxyl terminal (MIC6C) of MIC6 protein into the base of valine was performed by gene mutagenesis. The MIC6C W / V mutant gene fragment was amplified by PCR. / V / pGEX-4T-1 recombinant prokaryotic expression system, IPTG induced the expression of GST-MIC6C W / V mutant protein. The protein was used as a probe protein and Toxoplasma lysate GST sedimentation experiments, SDS-PAGE and Western blot analysis. Results: The gene fragment of MIC6C W / V mutant was obtained and the GST-MIC6C W / V mutant protein was prepared. No protein band was observed in the sedimentation product of MIC6C W / V mutant protein, while the MIC6C protein (unmutated protein) Of the product has a protein band, and can be anti-aldolase antibody recognition. CONCLUSIONS: MIC6 mutants were obtained in vitro; MIC6 mutants lost the property of aldolase.