人谷胱甘肽硫转移酶A1原核表达系统的构建及高效表达

High-effective experssion and construction of an prokaryote expression vector pET30a-glutathione-S-transferase A1 gene from human liver cDNA library and sequence determination

目的 对人谷胱甘肽硫转移酶A1(GSTA1)原核表达系统的构建及高效表达。方法 采用RT -PCR方法将肝组织中提取总RNA扩增人GSTA1基因的cDNA序列 ,将其插入到原核表达载体pET30a多克隆位点中 ,构建重组表达质粒 ,并用PCR扩增、酶切分析及序列测定等方法对重组质粒进行鉴定 ,并进行了高效表达。结果 人GSTA1克隆到原核表达载体pET30a多克隆位点中 ,测序结果同GenbankGSTA1(GI:2 2 0 914 5 3)cDNA序列相比较 ,在 5 12位点T→C ,氨基酸由Met→Thr,同源性为 99% ,基因登陆号为AY5 32 92 8。通过IPTG诱导表达 ,在 34.4KDa处 1、2、3、4小时的表达量分别为 4 1.8%、6 8%、6 8.3%、83%。结论 经Westernblot分析 。

Objective\ To express highly effective on IPTG-induced and construct an prokaryote expression vector carrying human glutathione-S- transferase (GST) A1 gene and to determine partial sequence analysis. Methods The GSTA1 cDNA was amplified and extracted from human liver total RNAs by RT-PCR approach and recombined with prokaryote expression vector pET30a.The recombined vector pET30a-GSTA1 was verified using PCR, restriction analysis and sequencing determination and induced with IPTG in 35℃. Results Human GSTA1 gene was recombined corrected with pET30a ,compared with Genbank , in code 512 T→C, amino acid Met→Thr;alignment core is 99%. The expressed fusion protein,with molecular weight of about 34.4KDa,were about 41.8%(1h),68%(2h),68.3%(3h),83%(4h)of the total cell protein by SDS-PAGE.Conclusion The protein expression of GSTA1 was demonstrated by Western blotting,it is correct.