Investigation Of Neisseria Gonorrhoeae: Synthetic Peptide Derived From An Epitope On PIB And Construction Of Prokaryotic Vector (pGEX-PIII)

Neisseria gonorrhoeae is etiological agent of gonorrhea, and the gonorrhea, nowadays, is one of sex transmitted diseases with the highest incidence rate at home and abroad. Mankind is the only natural host to the pathogen, mainly transmitting by sex contact. After infecting the Neisseria gonorrhoeae, the sufferers take place the damages of the genitourinary tract system, appearing urethritis, cervicitis, epididymitis, pelvic inflammatory disease, sterility and ectopic pregnancy. The diagnosing of gonorrhea is main basis on clinical performance and laboratory check, and the latter is the importance diagnose basis.Currently the common laboratory detection for Neisseria gonorrhoeae are secretion directly smear and culture. Final diagnosis must be depended on culture because chronic sufferers and women often have no typical symptoms, and the positive rate of smear without typical case is not high. The Neisseria gonorrhoeae is tender so it is not easy to develop, and needing special medium then can develop. It is normal to using selective medium. The specimen carrying makes its vigor descend and may also result in the mistake identification or leak check. For long time, many scholars look for a kind of simple, direct and fast detection method for Neisseria gonorrhoeae. For the past few years, people at home and abroad have been applied nucleic acid probe technique,polymerase chain reaction and ligase chain reaction into the detection of the Neisseria gonorrhoeae, but these methods request an equipments condition high and because grass-roots hospital currently still have no good quality control and assurance, can't be a gonorrhea diagnosis basis, the application is more and greatly limited. The immunology detect methods, such as euzymelinked immunosorbent assay, immunofluorescence and polyclonal antibody coagglutination, also hope to become a new kind of utility, control easily and operate easily detect means. But this kind method is depended on sieving of the valid antigen of Neisseria gonorrhoeae.The main antigen composition of Neisseria gonorrhoeae contains pilus, lipopolysaccharide and outer membrane protein. The lipopolysaccharide has poison and part of likeness with host carbohydrate in addition to higher variation same as the pilus, and these factors hindered them to become the valid composition in detection reagent for Neisseria gonorrhoeae. The outer membrane protein includes PI, PII and PIII. The PI with higher stability and immunogenicity is a main outer membrane protein, occupied 60% outer membrane protein total weight. The PI has two main types, PIA and PIB. It is to see frequently that infection caused by Neisseria gonorrhoeae expressing the PIA don't happen in the gential tract. After a long-term study to the PIB, Butt and Fletcher discovered that monoclonal antibody SM198 can react to all tested Neisseria gonorrhoeae strains expressing the PIB. Epitope discriminated by SM198 locates on PIB, five continuous amino acids of which present a higher conservatism, and the sequence is defined the minimum discriminating epitope for the SM198. Though the structure variation makes the PI produce serotype specificity, but the conservative antigenic determinant provides a latent target with reaction for antibody. The PIII is also one of the envelope antigens of the Neisseria gonorrhoeae, expressing stably on surface in all Neisseria gonorrhoeae strains. Be different from the PI, PII and pilus, the PIII that expressed only one type in all Neisseria gonorrhoea shows very high conservative. At home and abroad the research report about PIII is not much, maybe this is concerned with that PIII lose opportunity of becoming the candidate for vaccine because the antibody induced by PIII inhibit bactericidal action of other antibodies and complements. But because of high conservative the PIII can be made use of to develop the detection reagent for Neisseria gonorrhoeae.To obtaining complete antigen LD1-KLH, we synthesize a polypeptide LD1 that its sequence is corresponding to the epitope region of PIB of Neisseria gonorrhoeae with artificial synthetic peptide technique and then couple it to keyhole limpet hemocyanin with chemistry method. We immunize animal with the complete antigen to study its immunogenicity, and prepare LD1-KLH polyclone antiserum then detect its specificity. These will establish experiment bases to develop the correlated detection reagent using in clinical rapid diagnosis. Moreover, we construct correctly prokaryotic expression plasmid containing PIII protein of Neisseria gonorrhoeae to do some prepare in expressing artificially the protein for future. Ii is our hope that PIII protein can be made use of in studyof antigenicity and immunogenic, and also can establish experiment bases to develop the correlated detection reagent.The study were divided into two parts:Part one Investigation of synthetic peptide derived from an epitope on the outer membrane protein of Neisseria gonorrhoeae1. Analysis of the amino acid sequence of the epitope on the PIB of Neisseria gonorrhoeaeThe amino acid sequences of the epitope on the PIB of 18 strains from GenBank were analyzed by using the program DNAMAN. Identity of a sequence composed of 5 amino acids from the domain of the epitope on PIB of these 18 strains is 100%. Comparison of the inferred amino acid sequence of the domain, which is composed of 15 amino acids, containing the epitope of gonococcal strain R10 with those of other strains from GenBank, and the identity is above 80%. A peptide LD1 corresponding to the domain, which is composed of 15 amino acids, containing the epitope of gonococcal strain R10 was synthesized.2. Preparation of polyclonal antibody against LD1-KLHA peptide LD1, containing 15 amino acids, was synthesized corresponding to the sequence of the epitope on the PIB from gonococcal strain R10. LD1 was coupled to a carrier protein (KLH), and both the synthesis of LD1 and LD1-KLH were responsible by Xi'an Langene Bioscience co.,ltd. Both two rabbits and two guinea pigs were immunized with the LD1-KLH conjugate. A solution of the relevant antigen was diluted to two concentration of 200μg/ml and 100μg/ml, emulsified with an equal volume of Freund's complete adjuvant and a total volume of 1ml was injected respectively on rabbits and guinea pigs. Booster immunizations with the same antigen in Freund's incomplete adjuvant were given on days 15, 30 and 45 and blood was withdrawn on day 55. The immune sera were conserved in -20℃, and the preimmune sera were withdrawn before the immunization would be used as the negative control.3. Specificity of polyclonal antibody against LD1-KLH(1) Enzyme linked immunosorbent assay (ELISA): Immune sera produced following immunization were detected by ELISA. Every hole of ELISA plates were coated by incubation with 100μl LD1-KLH conjugate at a concentration of 25μg/ml. Dilution of immune serum of each animal engaged in reaction was increased by 2 times for plate 1 to 10. The result indicated that absorbance value decreased as the increase of antisera, and the titer of rabbit immune serum was between 1:3200 and 1:6400, and the titer of guinea pig immune serum was between 1:25600 and 1:51200.The anti-LD1-KLH serum can specifically recognize and react with the LD1-KLH conjugate.(2) Western blot: Gonococcal strains were splited by sample loading buffer. Immunological reactivity was also detected by Western blotting following separation of whole-cell lysate of Nesseria gonorrhoeae by 12% SDS-PAGE. The specificity of reactivity of both rabbit and guinea pig antiserum was investigated by Western blot, and both antisera produced a single band corresponding to the position of protein, of which the molecular weight is 37300.(3) Cross reaction of polyclonal antibody against LD1-KLH with different strains including Neisseria gonorrhoeae.The reactivity of both rabbit antiserum and guinea pig antiserum with Neisseria gonorrhoeae, Staphylococcus aureus,α-hemolytic streptococcus,β-hemolytic streptococcus, Streptococcus pneumoniae, Escherichia coli and Candida albicans was detected by ELISA. The result indicated that both rabbit antiserum and guinea pig antiserum only recognized and reacted with Neisseria gonorrhoeae, and no reaction was detected with the other strains.Part two Prokaryotic vector (pGEX-PIII) of Neisseria gonorrhoeae: Construction and appraisalUsing PIII sequence of Neisseria gonorrhoeae from GenBank as template, we designed and synthesized a pair of primers. The purpose DNA fragment was acquired from clinical strain genome of Neisseria gonorrhoeae by PCR technique. PCR products, after extracted with the mixture of hydroxybenzene, chloroform and isoamyl alcohol, were purified in the process of ethanol sedimentation. After the purified products and plasmid vector pGEX-3X were digested with restriction endonuclease BamH I and EcoR I, the larger fragments were reclaimed after 1% agarose electrophoresis. The larger fragments from pGEX-3X and PIII DNA were ligated by T4 ligase. Ligation product was used to convert competent DH5α. Positive colonies were selected after being cultivated on the solid medium containing ampicilli, digested with double enzymes and sequenced. The BLAST analysis was used for the sequenced results, the sequence of PIII and amino acid of Neisseria gonorrhoeae from the gene library. The DNA sequence demonstrated that the purpose DNA was agreeable to the one (strain FA1090) recorded in the GenBank, indicating that there was 99% identity for nucleotide, and 100% identity for the inferred amino acid.Conclusions:There is no studies reported in China about investigation of synthetic peptide derived from epitopes on the outer membrane protein PIB of Nesseria gonorrhoeae, and using it and PIII for preparation of gonococcal rapid diagnosis agent. In this study, we employed synthetic peptide for preparing of polyclonal antibody against outer membrane protein PIB of Neisseria gonorrhoeae. Antisera produced following immunization were detected by ELISA and Western blot. The result indicated that the synthetic peptide is immunogenic and has ability to induce a production of antibody, which specifically recognize and react with the epitope. The cost of development and production of outer membrane protein PIII would be a bill of huge numbers, if we employed peptide synthesis technology, because of the number of PIII amino acid sequence is about 20 times as many as the epitope on PIB. So we we employed molecular biological technology to successfully construct the Prokaryotic vector of the major outer membrane protein of Neisseria gonorrhoeae pGEX-PIII for large preparation of protein PIII. In a word, this study provided new candidates for development of a rapid diagnostic agent for detecting Neisseria gonorrhoeae.