Construction And Expression Of The Prokaryotic Recombinant Plasmid Containing The Nspa Gene Of Neisseria Gonorrhoeae

ObjectivesTo prepare immune serums aginst Neisseria gonorrhoeae which will be applied to detect the expression of recombinant protein NspA in Western Blot. Construct the prokaryotic expression system of nspA gene of Neisseria gonorrhoeae and induced it expressed in E. coli BL21(DE3) and provide a basis for further study of NspA.MethodsPreparation of immune serums against Neisseria gonorrhoeae:Two kinds of immunogen which inactivated Neisseria gonorrhoeae and Neisseria gonorrhoeae lysate were prepared, immune serum after immunizing rabbits with two kinds immunogen respectively were collected.Construction the prokaryotic expression system of pGEX4T-1-nspA and inducing the NspA fusion protein expressed:A pair of primers were designed according to the known sequence of nspA gene from Gen Bank. The fragment of nspA gene was amplified from genomic DNA of Neisseria gonorrhoeae by polymerase chain reaction (PCR), and PCR product was cloned into pGEX4T-1 which containing GST gene to construct the prokaryotic expression system of pGEX4T-1-nspA. The recombinant plasmid of pGEX4T-1-nspA was analyzed with restriction-endonuclease digestion, PCR and DNA sequencing analysis. The verified recombinant was transformed into E. coli BL21(DE3) and express by isopropy-β-D-thiogalactoside (IPTG) induced. The fusion protein GST-NspA was examined with SDS-PAGE and Western blot.ResultsImmune serums of gonococcus were prepared successfully.The titers of two kinds of immune serum were reached more than 1:12800. and a specific binding band appear in 44KDa position after these serums were used to detect the expression of recombinant protein NspA in Western Blot.The prokaryotic expression system of pGEX4T-1-nspA was constructed and induced to express the NspA fusion protein successfully:The fragment of nspA gene of Neisseria gonorrhoeae was amplified from genomic DNA of Neisseria gonorrhoeae by polymerase chain reaction; The prokaryotic expression system of pGEX4T-1-nspA with Neisseria gonorrhoeae nspA gene was constructed successfully by restriction-endonuclease digestion, PCR and DNA sequencing analysis verified; By the analysis of SDS-PAGE and Western blot, the recombinant NspA (rNspA), of Neisseria gonorrhoeae was expressed in prokaryotic expression system successfully, the fusion protein GST-NspA expressed is about 44kDa.ConclusionsImmune serums of gonococcus were prepared successfully, the immune serums can specifically recognize and combine with NspA recombinant protein. The results provided a basis for this study and further study of gonococcus:The prokaryotic expression system of pGEX4T-1-nspA was constructed and induced the NspA fusion protein to express in E. coli BL21(DE3) successfully. To provide a basis for the study biological activity of the NspA protein of Neisseria gonorrhoeae, animal immune experiment, the detection of Neisseria gonorrhoeae and the research of mucosal immune vaccine of Neisseria gonorrhoeae.