Construction Of Neutralizing Epitope Of IgA Protease Protein Of Neisseria Gonorrhoeae Expression Plasmids And Study Of Its Mucosal Immune In Mice

Objective:(1)To construct the recombinant plasmid of procaryotic expression pQE30-iga neutralizing epitope containing the neutralizing epitope of IgA protease protein gene of N. gonorrhoeae and to express fusion protein in E.coli, then purify it and analyse its immunogenicity.(2)To construct the recombinant plasmid of eukaryotic expression pcDNA3.1(-)-iga neutralizing epitope containing the neutralizing epitope of IgA protease protein gene of N. gonorrhoeae and to express fusion protein in mouse. To provide experimental basis for developing effective nucleic acid vaccines resisting the gonorrhea by inducing mucosal immunity in mouse using chitosan/ pcDNA3.1(-)-iga neutralizing epitope.Methods:(1)Expression, purification, renaturation and immunogenicity of neutralizing epitope of IgA protease protein Primers were designed by primer 5.0 software according to between from 1 601 to 2 722 of the IgA protease gene sequence of N. gonorrhoeae MS11. Polymerase chain reaction(PCR) was used to amplify the neutralizing epitope gene. After cleavaged by endonucleases, the fragment was subcloned to pQE30 procaryotic expression vector by linking reaction to generate recombinant plasmid pQE30- iga neutralizing epitope. The recombinant plasmid was inducted into E. coli M15 by IPTG after sequencing. The expressed production was analyzed by SDS-PAGE. The fusion protein was purified by Ni agar gelatin FF, and after degenerated by 8M Urea, the purified protein was analyzed by SDS-PAGE and Western-Blot. Then the pure inclusion protein was renaturated by concentration gradien dialysis of Urea, and the activited fusion protein was mixed with adjuvant to immune New Zealand White rabbits by intradermal injection. After coating wells with renaturated protein, the titers of neutralizing epitope in positive rabbit serum were detected by indirected-ELISA.(2)Preparation of Chitosan/Eukaryotic Expression plasmid containing the neutralizing epitope of IgA protease protein and mucosal immune to mouse Primers were designed by primer 5.0 software according to between from 1 601 to 2 722 of the IgA protease gene sequence of N. gonorrhoeae MS11. Polymerase chain reaction(PCR) was used to amplify the neutralizing epitope gene. After cleavaged by endonucleases, the fragment was subcloned to pcDNA3.1(-) eukaryotic expression vector by linking reaction to generate recombinant plasmid pcDNA3.1(-)-iga neutralizing epitope. After sequencing, chitosan/ pcDNA3.1(-)-iga neutralizing epitope nanopaticles was prepared and administered into 4 experimental mouse on weeks 1,3 and 5 by intravaginal, whereas chitosan / pcDNA3.1(-) and chitosan without DNA was given to control. Serum and vaginal washes samples collected every two weeks were used to determine anti iga neutralizing epitope IgA and IgG response by ELISA. Vaginas were moved from mice on day 5 and paraffin section was prepared for immune fluorescence to studing expression of the antigen of neutralizing epitope DNA vaccine in vivo.Results:The sequence analysis result showed that the target fragment were neutralizing epitope gene, according to the sequence reported by GenBank. A fusion protein was obtained after it was expressed in E. coli, and it was existed as insoluble inclusion bodies (IBs). After purificated with Ni agar gelatin FF, the protein was lysised by Urea. Western-Blot result proved that the recombinant protein can specifically react with monoclonal antibody labeled 6-His. The titers of neutralizing epitope antibody in positive rabbit serum were detected by ELISA and the highest titer was 1:6 400. Anti-iga neutralizing epitope IgA antibody response could be detected in serum and vaginal washes. Since day 4 after given plasmid chitosan/pcDNA3.1(-)-iga neutralizing epitope, faint fluorescence detected in some epithelial cells of vaginas indicated DNA expression in the tissues.Conclusion:(1)It was successful to construct the recombinant plasmid of procaryotic expression pcDNA3.1(-)-iga neutralizing epitope. We attained purified renaturation iga protein showed excellent immunoreactivity. (2)It was successful to construct the recombinant plasmid of eukaryotic expression pcDNA3.1(-)-iga neutralizing epitope. Vaginal immunization with chitosan/pcDNA3.1(-)-iga neutralizing epitope nanoparticles could induce mucosal immune response in genital tract. Inducing mucosal immunity would provide experimental basis for effective DNA vaccines resisting the gonorrhea.(3)Neutralizing epitope protein of IgA protease protein can express in vagina epithelial tissue of mouse.