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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 6R抗体和进行原位分子杂交研究创造了条件
Objective To obtain the gene of rat IL 6 receptor (IL 6R) and express it efficiently. Methods The sequence encoding IL 6R gene was obtained from the cDNA library of normal adult rat brain by PCR. After sequencing, the full length of IL 6R gene and the coding sequence of carboxyl terminal region of IL 6R gene were constructed respectively by PCR amplification and gene recombination, and then were introduced into E. coli DH5α to express the recombinant fusion protein by IPTG. Purified glutathione sepharose column of the expressed fusion protein encoded by the carboxy-terminal sequence. Results The gene of IL 6R (98-1493) in normal adult rats was obtained. The sequencing result was consistent with the published gene sequence. Recombinant proteins are expressed as inclusion bodies. SDS PAGE analysis showed that there were 1 specific protein bands at the relative molecular mass (Mr) of 740 0 0 and 430 0 0 respectively. High purity fusion protein was obtained after denatured, refolded and purified the inclusion body of carboxyl terminal gene. Conclusion The successful cloning of IL 6R gene in normal adult rat and its high expression in E.coli DH5α have created the conditions for the further preparation of anti-IL 6R antibody and in situ hybridization