阴道毛滴虫AK基因原核表达质粒的构建及在大肠埃希菌中的表达

Construction of Prokaryotic Expression Plasmid and Expression in E. coli of AK Gene of Trichomonas Vaginalis

目的构建阴道毛滴虫氢化酶体腺苷酸激酶(adenynate kinase,以下简称AK)基因原核表达质粒并予以表达。方法AKDNA的克隆载体PMD-18T-AK经限制性内切酶BamHI和XbaI双酶切,得到含这2个酶切位点之AKDNA片段,T4连接酶连接到含有2个酶切位点的原核表达载体PUC18,构建出重组原核表达质粒PUC18-AK,经PCR、酶切鉴定。重组质粒PUC18-AK经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,在大肠埃希菌中进行初步表达。结果AK基因体外扩增产物大小均为690bp,重组原核表达质粒经PCR及酶切鉴定表明获得正确重组子。在大肠埃希菌中表达出AK蛋白,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析与理论预测值相符。经蛋白质印迹法(Western blotting)鉴定具有免疫原性。结论成功地构建了AK基因原核表达质粒,并在大肠埃希菌中表达了AK蛋白。

Objective To construct and identify prokaryotic expression plasmid of the hydrogenosomal adenylate kinase of triehomonas vaginalis. Methods The cloning vectors（ containing full length of AK DNA and two restriction sites： BamHI and XbaI）were first cut by two restriction enzyme： BamHI and XbaI, and the same as the prokaryotic expression plasmid; then AK DNA and the digested vector were ligated by T4 DNA ligase, and recombinant prokaryotie expression vector was formed. It was certified by PCR and restriction analysis. The compound gene PUC18 - AK was expressed in E. coli by IFFG. Results The size of amplified AK gene was 690 bp. The correct recombinant plasmid PUC18 - AK was isolated and confirmed by PCR and restriction analysis. The expressed protein AK in E. coli was identified by SDS - PAGE and western blotting. Conclusion The prokaryofic expression plasmid of AK gene was successfully constructed, which was then expressed in E. coli.