CAJ | 학술논문

目的探讨克隆和表达沙眼衣原体热休克蛋白60(hsp60)基因.方法 PCR分离扩增hsp60的基因片段,纯化后双酶切,克隆到原核表达载体pET-28a,构建重组表达载体pET-28a-hsp60.PCR、双酶切及测序鉴定.转染大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE、Western印迹检测.结果 PCR与双酶切结果显示所构建的重组质粒已成功地克隆hsp60基因,测序结果与基因库公布的一致.SDS-PAGE检测表达产物.在相对分子量60 000处有表达条带.Western印迹鉴定是表达目的蛋白.纯化后纯度达90%以上,产量为17.85 mg/L.结论构建pET-28a-hsp60重组体并成功表达可溶性hsp60蛋白.
목적 탐토극륭화표체사안의원체열휴극단백60(hsp60)기인.방법 PCR분리확증hsp60적기인편단,순화후쌍매절,극륭도원핵표체재체pET-28a,구건중조표체재체pET-28a-hsp60.PCR、쌍매절급측서감정.전염대장간균BL21(DE3),IPTG유도표체,SDS-PAGE、Western인적검측.결과 PCR여쌍매절결과현시소구건적중조질립이성공지극륭hsp60기인,측서결과여기인고공포적일치.SDS-PAGE검측표체산물.재상대분자량60 000처유표체조대.Western인적감정시표체목적단백.순화후순도체90%이상,산량위17.85 mg/L.결론 구건pET-28a-hsp60중조체병성공표체가용성hsp60단백.
Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.