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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 express the adenovirus vector of adenovirus of Trichomonas vaginalis. Methods The cloned vector PMD-18T-AK of AKDNA was double-digested with restriction enzymes BamHI and XbaI to obtain the AKDNA fragment containing the two restriction sites. The T4 ligase was ligated into the prokaryotic vector containing two restriction sites The expression vector PUC18 was constructed, and the recombinant prokaryotic expression plasmid PUC18-AK was constructed and identified by PCR and restriction enzyme digestion. The recombinant plasmid PUC18-AK was induced by isopropyl-β-D-thiogalactoside (IPTG) and expressed in Escherichia coli. Results The size of AK gene in vitro was 690bp. The recombinant prokaryotic expression plasmid was identified by PCR and restriction enzyme digestion. The AK protein was expressed in Escherichia coli and the SDS-PAGE analysis was consistent with the theoretical predictions. Western blotting was used to identify the immunogenicity. Conclusion The prokaryotic expression plasmid of AK gene was successfully constructed and AK protein was expressed in Escherichia coli.