大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2的表达、纯化及抗体制备

Expression and Purification of SERPINB2 and Preparation of its Polyclonal Antibody

目的:原核表达、纯化大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2(SERPINB2),并制备其多克隆抗体。方法:设计扩增大鼠Serpinb2全长基因的特异引物,通过PCR扩增出该基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果:原核表达了大鼠Serpinb2全长基因,纯化了SERPINB2蛋白,并获得了抗SERPINB2的多克隆抗体,抗体效价达到1:25600,Western印迹结果显示该抗体能特异识别原核及真核细胞表达的SERPINB2。结论:SERPINB2能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究SERPINB2的功能奠定了基础。

Objective：To express and purify serine（or cysteine） proteinase inhibitor B2（SERPINB2） in E.coli,and prepare the polyclonal antibody against SERPINB2.Methods：The specific primers were used to amplify full length of Serpinb2 gene by PCR,then the gene was inserted into the vector pGEX-KG containing GST gene,GST-SERPINB2 fusion protein was expressed in E.coli,and purified by using Glutathione Sepharose 4B.Then anti-SERPINB2 antiserum was prepared in mice,the titer was determined by ELISA and the specificity was identified by Western blot.Results：Serpinb2 was expressed and SERPINB2 protein was purified,the polyclonal antibody against SERPINB2 was prepared,the titer of this antibody was about 1∶25 600,and SERPINB2 expressed in prokaryotic and eukaryotic cells can be recognized by this antiserum.Conclusion：SERPINB2 protein can induce the effective and specific polyclonal antibody in mice,it is the basis to understand the function of SERPINB2