Construction, Expression And Identification Of Prokaryotic And Eukaryotic Expression Vectors Of Neisseria Gonorrhoeae PⅠB Gene

Objectives:To construct prokaryotic expression vector pET30a/Pâ… B and eukaryoticexpression vectors pcDNA3.1(-)/Pâ… B of Neisseria gonorrhoeae (NG) and toexpress the recombinant protein in E.coli BL21 and HeLa cells respectively. Thiswill be very helpful for the further research of Pâ… B immune functions, thepreparation of antibody and the new vaccine development.Methods:According to the Pâ… B gene sequence of NG MS11 strain, a pair of primerswere designed and synthesized. Polymerase chain reaction (PCR) was used toamplify the Pâ… B gene. PCR products were purified and cloned into the prokaryoticexpression vector pET30a(+). The recombinant plasmids pET30a/Pâ… B confirmedby PCR, restriction endonuclease digestion and sequencing was transformed intoE.coli BL21. IPTG was added to induce the expression of the protein. The expressedprotein was identitied by SDS-PAGE and Western blot. Besides, the recombinationeukaryotic expression vectors, pcDNA3.1(-)/Pâ… B was constructed. Then therecombinate expression plasmids were transfected into Hela cells in vitro usingliposome. The expression products were analyzed by immunocytochemistry.Results:1. The Pâ… B gene was inserted into pET30a(+) vector to construct pET30a/Pâ… Brecombinant. The results of restrict emzyme digestion, PCR and sequencingindicate the inserted sequence was conformed to Pâ… B gene sequence of NGBL21 strain completely. 2. The pET30a/Pâ… B recombinant was transformed into E.coli BL21 and proteinexpression was induced by adding IPTG. The target protein bands wereobserved obviously both on SDS-PAGE gel and in Western blot analysis. Themolecular weight of the expressed protein is similar to the deduced value.3. The recombinate expression plasmids pcDNA3.1(-)/Pâ… B were transfected intoHela cells in vitro using liposome. The expression of Pâ… B has been detected byimmunocytochemistry.Conclusion:1. Prokaryotic expression vector pET30a/Pâ… B was constructed successfullyand it can express target protein in E.coli.2. Eukaryotic expression vector pcDNA3.1(-)/Pâ… B was constructedsuccessfully and it can express target protein in eukaryotic cells.