大鼠肝再生增强因子编码区的cDNA克隆和原核表达

The cDNA clone of the coding region of rat augmenter of liver regeneration gene and its prokaryotic expression

目的：克隆大鼠肝再生增强因子（ＡＬＲ）编码区ｃＤＮＡ并进行原核表达。方法：提取新生大鼠肝脏总ＲＮＡ为模板，以寡聚ｄＴ为引物逆转录合成第一链ｃＤＮＡ，再以我们设计的ＰＣＲ引物进行ＰＣＲ扩增获取大鼠肝再生增强因子编码区双链ｃＤＮＡ；以基因重组技术构建大鼠ＡＬＲ的原核表达质粒，然后将重组表达质粒转化至大肠杆菌内以ＩＰＴＧ诱导表达。结果：成功克隆出大鼠肝再生增强因子编码区ｃＤＮＡ，其序列与以前报道的完全一致；构建的大鼠ＡＬＲ原核表达重组质粒在大肠杆菌内高效表达ｒＡＬＲ融合蛋白，其表达量占菌体蛋白３０％。结论：大鼠肝再生增强因子编码区ｃＤＮＡ的克隆及其在大肠杆菌的高效表达为ＡＬＲ的深入研究奠定了基础。

Objective: To clone the coding region cDNA of rat augmenter of liver regeneration (rALR) and to express it in prokaryotic cells. Methods: Total RNA of new born rat liver was extracted as a template, first strand of cDNA was synthesized by reverse transcription with olig(dT)-primer and then amplified by PCR with the primers designed by ourselves. The rALR cDNA identified by gene sequencing was cloned into plasmid pET32a by means of recombinant gene technology. The recombinant plasmid pET32a-rALR was transformed into E.coli BL21 (DE3) and recombinant rALR fusion protein was expressed under the induction of IPTG. Results: The cDNA containing integrity coding region of rALR was obtained, and its sequence was in correspondence with that reported. The rALR fusion protein was expressed with high efficiency in E.coli BL21 (up to 30% of total bacterial soluble protein) with the recombinant plasmid pET32a-rALR constructed. Conclusion:The cDNA clone of rALR and the expression of rALR protein will be helpful for the advanced study of ALR.