CAJ | 학술논문

目的：构建含有与溶酶体膜蛋白2（LAMP-2）P41-49完全同源序列的尿路致病性大肠杆菌（UPEC）Ⅰ型菌毛FimH1-156为目的基因的原核载体，表达并纯化FimH1-156融合蛋白。方法采用PCR克隆pPKL241质粒获取 FimH1-156基因，插入原核表达载体pET28a（＋）；将重组表达质粒转染感受态 E ．coli BL21（DE3），经IPTG诱导表达 FimH1-156融合蛋白，超声裂解细菌，Ni-NTA层析法进行纯化，聚丙烯酰胺凝胶电泳（SDS-PAGE）分析、蛋白免疫印迹法（Western blot）对其进行鉴定。结果成功构建表达载体，经对融合蛋白表达条件的优化，在IPTG 浓度为1 mmol/L ，诱导4 h目的蛋白表达量最高，主要以包涵体形式存在；Western blot证实该FimH1-156融合蛋白可与抗6× His单克隆抗体发生结合反应。结论成功克隆FimH1-156融合蛋白表达基因并构建至原核表达载体，获得了包涵体纯化的Fim H1-156融合蛋白，为下一步建立实验动物模型奠定了基础。
목적：구건함유여용매체막단백2（LAMP-2）P41-49완전동원서렬적뇨로치병성대장간균（UPEC）Ⅰ형균모FimH1-156위목적기인적원핵재체，표체병순화FimH1-156융합단백。방법채용PCR극륭pPKL241질립획취 FimH1-156기인，삽입원핵표체재체pET28a（＋）；장중조표체질립전염감수태 E ．coli BL21（DE3），경IPTG유도표체 FimH1-156융합단백，초성렬해세균，Ni-NTA층석법진행순화，취병희선알응효전영（SDS-PAGE）분석、단백면역인적법（Western blot）대기진행감정。결과성공구건표체재체，경대융합단백표체조건적우화，재IPTG 농도위1 mmol/L ，유도4 h목적단백표체량최고，주요이포함체형식존재；Western blot증실해FimH1-156융합단백가여항6× His단극륭항체발생결합반응。결론성공극륭FimH1-156융합단백표체기인병구건지원핵표체재체，획득료포함체순화적Fim H1-156융합단백，위하일보건립실험동물모형전정료기출。
Objective To construct and express a prokaryotic expression vector carrying the gene of FimH 1-156 that comprises human lysosome membrane protein 2 P41-49 gene ,and to express and purify the fusion protein .Methods FimH1-156 gene was cloned from plasmid pPKL241 by PCR ,and inserted into vector pET-28a(+ ) to obtain prokaryotic expression plasmid pET-28a-FimH . After transforming Escherichia coli BL21(DE3) with pET-28a-FimH ,fusion protein FimH1-156 was expressed under induction .The target fusion protein was purified ,and its antigenicity was detected through Western blot .Results The expressed recombinant pro-tein was purified ,the expression of protein was the highest when IPTG was 1 mmol/L and 4h after induction ,it was expressed as include body form ,and the expressed protein was identified to react with monoclonal antibodies 6 × His by Western blotting .Conclu-sion We cloned FimH1-156 fusion protein expressed genes successfully ,constructed prokaryotic expression vector ,and won the in-clusion body purification of FimH1-156 fusion protein .