重组C8orf32蛋白的表达、纯化及抗体制备

Expression,Purification and Antibody Preparation of Recombinant C8orf32 Protein

C8orf32是一种功能未知基因,其mRNA含量在乳腺癌组织中显著高于正常乳腺组织。将其开放式阅读框插入pGEX-6P1原核表达载体T7启动子控制下的GST编码基因下游,构建了C8orf32蛋白表达质粒pGEX-6P-C8。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达了GST-C8orf32融合蛋白。经带有GST标签的位点特异性蛋白酶切割除去GST-C8orf32融合蛋白的GST标签,获得了N端带有8个多余氨基酸残基的C8orf32蛋白,蛋白纯度为95%左右。N端氨基酸序列分析表明该蛋白N端氨基酸序列正确,质谱鉴定进一步证明所表达C8orf32蛋白的正确性。用制备的C8orf32蛋白免疫新西兰白兔,获得了能够正确识别C8orf32蛋白的抗血清。该蛋白及其抗体的成功制备,为进一步研究C8orf32蛋白的结构功能和体内分布打下了基础。

C8orf32 is a gene which has not been functionally characterized, the mRNA level of this gene is significantly higher in breast cancer tissues than that in normal breast tissues. The amplified cDNA fragment was inserted into the pGEX-6P1 vector fused with the upstream GST gene. The expression vector was transformed into the E. coli BL21 （ DE3 ） strain and expression of GST - C8orf32 fusion protein was induced by IPTG.. After removal of GST tag by site-specific protease,the C8orf32 protein fused with an eight amino acid peptide tag was obtained. The purity of recombinant C8orf32 protein was about 95%. The identity of the purified protein was confirmed by N-terminal sequencing and tandem mass spectrometry. The polyelonal antibody was prepared by immunizing the New Zealand white rabbits with C8orf32 protein. The polyclonal antibody was proved to recognize the C8orf32 protein correctly. The purified C8orf32 protein can be used for structural and functional studies and the polyclonal antibody can be used for tissue specific protein expression profiling.