大鼠IL-6受体基因的克隆及在大肠杆菌中的表达

Cloning of rat IL-6 receptor gene and its expression in E. coli

目的 获取大鼠IL 6受体 (IL 6R)的基因并高效表达。方法 用PCR技术 ,从正常成年大鼠脑的cDNA文库中 ,获得编码IL 6R基因的序列。测序后 ,通过PCR扩增和基因重组 ,分别构建IL 6R基因全长和羧基端部分编码序列的表达载体 ,并导入大肠杆菌DH5α中 ,通过IPTG诱导表达重组融合蛋白。对表达的羧基端序列编码的融合蛋白过谷胱甘肽琼脂糖柱进行纯化。结果 获得正常成年大鼠IL 6R(98～ 1493位 )的基因 ,测序结果与已发表的基因序列相一致。重组蛋白以包涵体的形式进行表达。经SDS PAGE分析 ,在相对分子质量 (Mr)为 740 0 0和 430 0 0处 ,各有 1条特异的蛋白带。对羧基端基因表达的包涵体形式的蛋白进行变性、重折叠及纯化后 ,得到了高纯度融合蛋白。结论 成功地克隆正常成年大鼠IL 6R基因 ,并在E .coliDH5α中高效表达 ,为进一步制备抗IL

Aim To obtain rat interleukin-6 receptor (IL-6R) gene and express efficiently in E. coli. Methods IL-6R gene were amplified by PCR from normal adult rat brain cDNA library. Expression vectors of whole IL-6R gene and its carboxyl terminal partial gene sequence were constructed by PCR and gene recombination technique. After sequencing, the vectors were transformed into E.coli DH5α and recombinant fusion protein was expressed via induction of IPTG. Fusion protein encoded by carboxyl terminal gene sequence was purified through glutathione agarose column. Results The sequence of cloned rat IL-6R gene was identical with that having reported. Expressed fusion proteins existed in the form of inclusion body. Two protein bands of Mr 74 000 and 43 000 appeared on SDS-PAGE gel. The protein encoded by carboxyl terminal gene sequence was denatured, refolded, and purified to a high purity. Conclusion The rat IL-6R gene was cloned successfully and expressed efficiently in E.coli DH5α, which creates a favourable condition for preparation of anti-IL-6R antibody and in situ hybridization analysis.