人Ⅱ型丙氨酸氨基转移酶表达载体的构建及在大肠杆菌中的表达

Cloning and expression of human alanine aminotransferase Ⅱ

目的 构建基因工程菌株 ,以获得重组人II型丙氨酸氨基转移酶 (ALT2 )蛋白。方法 逆转录PCR法从人肌肉组织总RNA中扩增ALT2基因片段 ,插入原核表达载体pET - 2 8a ,构建成融合表达质粒pET2 8 ALT2 ,将其转化大肠杆菌BL2 1DE3,IPTG诱导表达。SDS PAGE、活力测定、活性染色及westernblot鉴定表达产物。结果 重组质粒pET2 8 ALT2测序和酶切结果与预期完全符合。IPTG诱导后 ,阳性菌体裂解物上清ALT2活性很高 ,PAGE出现一分子量 5 80 0 0蛋白条带 ,可被抗ALT1抗血清识别 ,酶活性染色显示有ALT活性。结论 已成功将ALT2基因克隆到pET 2 8a载体 ,ALT2蛋白得到可溶性高效表达。

Objective To construct a recombinant plasmid for expression of human alanine aminotransferase Ⅱ (ALT2).Methods ALT2 cDNA was amplified by RT-PCR from total RNA of human liver,and cloned into pET-28a by restriction and subsequent ligation.Expression of ALT2 was induced by IPTG and demonstrated by SDS-PAGE,enzyme reaction and western blot.Results Sequencing and restriction analysis results indicated ALT2 gene was cloned in frame into pET-28a.Western blot showed a band with molecular weight of 58 kD reacted with anti-ALT1 serum.Enzyme dete rmination and activity staining in native PAGE indicated a high,soluble expression of ALT activity in the recombinant plasmid.Conclusion Human ALT2 gene was cloned and ALT2 protein with native activity expressed in satisfactory level.ALT2 shares some homology with ALT1.