摘要
目的用聚合酶链反应法（ＰＣＲ）和基因重组技术构建重组人肝再生增强因子原核表达载体并在大肠杆菌中进行表达。方法利用 ＰＣＲ方法从我们构建的ｐＵＣ１９－ｈＡＬＲ质粒中扩增出带Ｎｄｅｌ和 ＢａｍＨＩ酶切位点的ｈＡＬＲ编码区ＤＮＡ。ＰＣＲ产物和质粒ｐＥＴ１１ａ分别被相应内切酶消化，然后被Ｔａ ＤＮＡ连接酶连接以获取重组表达质粒。重组表达质粒ｐＥＴ１１３－ｈＡＬＲ经电转化人大肠杆菌ＢＬ２１（ＤＥ３）中，在ＩＰＴＧ诱导下表达ｒｈＡＬＲ。结果内切酶消化和ＤＮＡ测序证实ｈＡＬＲ的编码区正确地插人载体，重组ｈＡＬＲ在大肠杆菌中成功地表达。其分子量是１．５×１０４与预计的理论值相符合，表达量占菌体总蛋白的３０％；它不仅存在于破碎菌的上清液中，也存在于沉淀中。结论ｈＡＬＲ表达质粒的成功构建和重组ｈＡＬＲ在大肠杆菌中的成功表达，为进一步研究它的生物学功能和制备相应抗体奠定了基础。
Objective To construct prokaryotic expression vector of hALR and express rhALR in E.Coli. through PCR and recombinant gene technology. Methods The coding region with Nde 1 and BamH I sites of hALR was obtained from pUC19-hALR constructed with PCR method. PCR product and plasmid pET11a were digested by corresponding endonucleases, respectively. The fragments cut were ligated by T4 DNA ligase to gain recombinant expression vector. The recombinant plasmid PETI la-hALR was electrotransformed into BLZI(DE3) strain. The rhALR was expressed in the bacteria under induction of IPTG. Results Endonucleases digesting and DNA sequening confirmed that the coding region of hALR was correctly inserted into the vector. The rhALR was successfully expressed in E.Coli.. Its MW. with l.5 x 104 was in correspondence with theoretic value and its amount is 30% of total bacteria protein. It existed not only in supernatant but also in precipitation of broken bacteria. Conclusion The successes in construction of expressive vector of hALR and in expression of rhALR in E.Coli. make it possible to study further on its biological fuctions and antibody preparation.
出处
《中华肝脏病杂志》
CAS
CSCD
2000年第1期9-11,共3页
Chinese Journal of Hepatology
基金
国家自然科学基金
国家教委优秀青年教师基金
关键词
基因表达
肝再生增强因子
肝再生
大肠杆菌
Gene expressions
Genetic vectors
Augmenter of liver regeneration
