Animal Study On The Adhesional Inhibiton Of Polyclonal Anti-Porin I Antibody

Gonorrhea is at the present time one of the most common sexually transmitted diseases in the world. In our country the prevallance of gonococcal is increasing rapidly in recent decades. Gonorrhea is a purulent inflammation of the mucous membrane surface caused by a sexually transmitted microorganism, Neisseria gonorrhoeae. Virtually any mucous membrane can be infected. Due to the extensive transmission, gonorrhea seriously endangers mental and physical health of the mankind and social stability as well. Since Neisseria gonorrhoeae has throughout its history developed resistance to traditional antibiotics, it might be very important to develop an effective vaccine to prevent the infection of Neisseria gonorrhoeae.The pathophysiology of N gonorrhea and the relative virulence of different subtypes depend on the antigenic characteristics of the respective surface proteins. Certain subtypes are able to evade serum immune responses and are more likely to lead to disseminated (systemic) infection.The outer membrane of Neisseria gonorrhoeae includes a variety of protein, the most abundant of which is known as protein I (PI), occupying about 60% of total outer membrane protein. PI may function as a porin creating a hydrophilic diffusionchannel and allow uptake of essential nutrients. Unlike the other major surface antigens, pili, PII and LPS, PI dose not undergo antigenic variation during infection. PI may also play an important role in pathogenesis through its ability to interact with the cell membrane of eukaryotic cells, that means PI is related to the adherence of gonococcus to mucosa. PI is also a major target for immune attack on the gonococcus and can stimulate the production of opsonins. And anti-PI antibody can also activate complement-mediated bactericidal activity in mice. Thus, by virtue of its stability and antigenicity, PI is of considerable interest as a potential gonococcal vaccine candidate.The goal of our study is to investigate the blockness effects of purified polyclonal Anti- Porin I antibody on N.gonorrhoeae adherence to genitourinary tract epithelia of BALB/c mouse. Polyclonal Anti GST-PI antibody was generated by immunizing rabbit with GST-PI fusion protein which was constructed and expressed by ourselves. We established mice model of gonorrhea. In order to evaluate the effects of PI-IgG on gonococcus adhesion to vagina mucus, the macroscopic and pathological assessing as well as gonococcus culture was employed after gonococcus challenge on PI-IgG immunized mice. We hope our study may lead to a potential possibility for the further study of gonococcal vaccine.Methods1. Induction and purification of GST-PI fusion proteinThe expression recombinant (pGEX-4T-2/PI) was transformed to E.Coli P2392, then GST-PI fusion protein was induced by IPTG. The purification process was: SDS-PAGE, excision of 60 KDa protein band, and electroelution to collect GST-PI fusion protein.2. Preparation and detection of Polyclonal antibody seraFirst immunization: Each rabbit was inoculalted with emulsified immunizator containing GST-PI fusion protein and complet Fu's immunoadjuvant. Enhancingimmunization: Each was inoculated for further three times with the emulsified immunizator containing GST-PI protein and incomplete Fu's immunoadjuvant. On the fifth day, the antibodies potence value of rabbit sera was detected by doubleeagar immunodiffusion. Last immunization: Each was inoculated with PI protein without immunoadjuvant, and on the fifth day the blood of rabbit was collected and the sera were separated out.3. Purification of the polyclonal antibody of rabbit anti-PI seraThe purified immunolobulin G was obtained by ammonium sulphate deposition and DEAE cellulose chromatography. The titer of antibody were determined by doubleeagar immunodiffusion, and the purity were characterized by SDS-PAGE.4. Adhesion modelRoutinly degerm the cunnus of BALB/c mouse, centrifugate and callback the culture solution of Diplococcus gonorrhoeae, then inoculate the bacteria endovaginal (5×10⁸ bacteria per mouse). The vaginal mucosa and the secre...