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目的克隆铜绿假单胞菌外膜蛋白OprF基因,构建原核表达载体并鉴定表达。方法从铜绿假单胞菌中提取基因组DNA,PCR扩增OprF羧基端,克隆至原核表达载体pET28b中,构建重组表达载体pET28b-F。重组质粒经酶切和序列测定后,转化E.coliBL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,进行SDS-PAGE检测。经Ni-NTA亲和层析柱纯化后OprF蛋白免疫BALB/C小鼠,通过杂交瘤方法制备相应单克隆抗体并用ELISA方法进行鉴定。结果 pET28b-F原核表达载体构建成功。重组表达质粒经IPTG诱导后表达外膜蛋白OprF。获得4株高分泌型特异性单克隆细胞株,其中2株单克隆抗体可用于制备双抗体夹心法ELISA试剂盒,检测铜绿假单胞菌。结论成功克隆铜绿假单胞菌外膜蛋白OprF羧基端基因片段并在大肠杆菌中进行表达,筛选出特异性抗OprF单克隆抗体。
Objective To clone OprF gene of Pseudomonas aeruginosa outer membrane protein and construct prokaryotic expression vector and identify its expression. Methods The genomic DNA was extracted from Pseudomonas aeruginosa. The carboxyl terminal of OprF was amplified by PCR and cloned into prokaryotic expression vector pET28b to construct recombinant expression vector pET28b-F. The recombinant plasmids were transformed into E.coli BL21 and induced by IPTG after digestion and sequencing. The recombinant plasmids were induced by SDS-PAGE. BALB / C mice were immunized with OprF protein by Ni-NTA affinity chromatography. The corresponding monoclonal antibodies were prepared by hybridoma method and identified by ELISA. Results The prokaryotic expression vector pET28b-F was successfully constructed. The recombinant plasmid was induced by IPTG to express OprF. Four monoclonal cell lines secreting monoclonal antibodies were obtained, of which two monoclonal antibodies could be used to prepare double antibody sandwich ELISA kit for the detection of Pseudomonas aeruginosa. Conclusion The OprF carboxy-terminal gene fragment of Pseudomonas aeruginosa outer membrane protein was successfully cloned and expressed in E. coli, and the specific anti-OprF monoclonal antibody was screened out.