人Sec61β蛋白的原核表达及其多克隆抗体的制备

Prokaryotic Expression of Human Sec61β Protein and Preparation of Polyclonal Antibody against Sec61β

目的构建人Sec6β基因的表达载体并在大肠埃希菌中表达Sec61β蛋白并制备多克隆抗体，为后续研究Sec61β蛋白及其自身抗体在人大肠癌诊断中的价值奠定基础。方法用RT-PCR技术从人大肠癌组织中扩增出Sec61β基因，并与克隆载体pMD18-T连接转入XL1-BLUE克隆菌中，筛选出重组阳性菌落并提取质粒，用EcoR1、XholI将目的基因从重组克隆载体上切下并与同样双酶切后的表达载体pET-28a连接，转入表达菌BL21（DE3）中，IPTG诱导表达Sec61β重组蛋白，SDS-PAGE鉴定蛋白表达情况。从SDS-PAGE凝胶上切取目的蛋白免疫家兔，获得抗血清。经镍柱纯化后复性获得基因重组蛋白，以该蛋白为抗原，间接ELISA法测定血清抗体效价。Western印迹法检测抗体特异性。结果成功扩增出Sec61β基因（270bp），并诱导表达出Sec61β基因工程蛋白，大小约为15kD，以包涵体形式存在。制备的Sec61β多克隆抗体经Western印迹分析，可与人体组织中目的蛋白特异性结合。结论成功构建了Sec61β的原核表达载体并在大肠埃希菌表达了人Sec61β蛋白，通过SDS-PAGE凝胶的方法切取重组蛋白免疫家兔获得了特异性多克隆抗体。

To construct the expression plasmid containing Sec61 β, express See61β protein in E. Coli and prepare polyclonal antibody in an attempt to lay a foundation for further study of the role of See61β autoantibody in diagnosis of colorectal cancer （CRC） . Methods The object gene of Sec61β was obtained from human CRC tissues by RT-PCR. Then it was connected with plasmid pMD18-T and transfered to E. Coli XL1-BLUE for positive colony screening and plasmid extraction. Afterwards, Sec61β was digested from the recombinant plasmid by using EcoR I and Xho I, and was connected to the expression plasmid pET-28a which was also digested by the same enzymes. Then, it was transfered to E. Coli BL21 （DE3） . IPTG was used to induce therecombinant protein expression, and SDS-PAGE was conducted to detect the protein expression. Rabbits were immunized with the expressed protein obtained from the SDS-PAGE gel. The renatured recombinant protein purified from the Nickel column was used as the antigen. The titer of polyclonal antibody was measured by indirect ELISA. The specificity of See61β antibody was characterized by Western blotting. Results The Sec61β gene was successfully amplified and See61β protein was induced to express. Its molecular weight was 15kD and it mainly existed in inclusion bodies. Western blotting re- vealed that the prepared See61 β polyclonal antibody could specifically recognize the See61 β protein in tissues. Conclusion The prokaryotie expression vector of Sec61β was successfully constructed and the Sec61β protein expressed in E. Coli BL21 （DE3） . High titer polyclonal antibodies were obtained by immuning rabbits with Sec61β cut from SDS-PAGE gel.