肌球蛋白结合蛋白C的表达和鉴定

The expression and identification of myosin-binding protein C

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 establish an efficient method of expressing recombinant human myosin-binding protein C,fast type （MYBPC2）gene in Escherichia coli and to purify the protein by affinity chromatograph optimally.Methods：According to the mRNA sequence of MYBPC2,a pair of primers was designed,which was respectively added restriction enzyme cutting site of BamHI and Hind Ⅲ at 5＇terminus.The sequence was amplified corresponding to 284th and 579th amino acid residue of the full length protein and inserted into pMalc5e plasmaid after restriction Enzyme cutting.Sequencing the recombinant plasmid,then it was transformed into Escherichia coli.IPTG induced the expression of MYBPC2 fusion protein.Affinity chromatograph was used to purify the protein.The molecular weight and purity was roughly determined by SDS-PAGE.Measuring of protein concentration was to estimate protein yield.Animals were immunized with purified protein to obtain antiserum.Using commercial anti-MYBPC2 antibodies and antiserum to combine MYBPC2 fusion protein by Western blot.Results：The recombinant plasmid inserted MYBPC2 gene was constructed.Each liter of culture medium could produce 30mg purified fusion protein.This recombinant protein could combine with its antibodies and antiserum specifically.Conclusion：The MYBPC2-MBP fusion protein could be efficiendy synthesized and purified by this method,which present favorable antigen specificity and immunogenicity.