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目的:探讨优化原核表达方法以及用亲和层析的方法制备重组人快型肌球蛋白结合蛋白C(MYBPC2)。方法:根据MYBPC2的序列设计一对引物,在上下游引物的5’端分别加上BamHI和HindⅢ酶切位点。以健康人骨骼肌cDNA为模版,扩增第284位至第579位氨基酸残基对应的碱基序列,经双酶切后插入pMalc-5e载体中,测序鉴定重组质粒的序列,将构建好的重组质粒转化大肠杆菌中,IPTG诱导重组蛋白MYBPC2-MBP的表达,亲和层析纯化重组蛋白,蛋白电泳法鉴定蛋白的分子量及纯度,测定蛋白浓度并估算蛋白纯化后得率。用商品化的抗MYBPC2抗体证实所表达蛋白序列的正确性。以纯化的蛋白免疫动物获得动物免疫血清,用Western Blot的方法鉴定此蛋白的免疫原性。结果:人MYBPC2蛋白表达载体构建成功,每升大肠杆菌菌液可生产纯化后的重组MYBPC2蛋白约30mg,Western Blot显示重组蛋白与其抗体及抗血清特异结合。结论:本方法可以高效制备生产人MYBPC2蛋白片段,具备良好的抗原特异性和免疫原性。
OBJECTIVE: To investigate the optimization of prokaryotic expression and the preparation of recombinant human myoblast-binding protein C (MYBPC2) by affinity chromatography. Methods: According to the sequence of MYBPC2, a pair of primers was designed. BamHI and HindIII restriction sites were added to the 5 ’end of the upstream and downstream primers, respectively. Using the cDNA of healthy human skeletal muscle as a template, the base sequence corresponding to the amino acid residues from the 284th to the 579th was amplified, inserted into pMalc-5e vector after double enzyme digestion, and sequenced to identify the sequence of the recombinant plasmid. The constructed Recombinant plasmids were transformed into Escherichia coli. IPTG induced the expression of recombinant protein MYBPC2-MBP. The recombinant protein was purified by affinity chromatography. The molecular weight and purity of the recombinant protein were identified by protein electrophoresis. The protein concentration was determined and the protein yield was estimated. The correctness of the expressed protein sequence was confirmed by the commercial anti-MYBPC2 antibody. Animals were immunized with purified protein to obtain animal immune sera. The immunogenicity of this protein was identified by Western Blot. Results: The human MYBPC2 protein expression vector was constructed successfully, and about 30 mg purified recombinant MYBPC2 protein was produced per liter of E. coli bacteria. Western Blot showed that the recombinant protein specifically bound with its antibody and antisera. Conclusion: This method can efficiently produce human MYBPC2 protein fragment with good antigen specificity and immunogenicity.