The Major Outer Membrane Protein (PⅠA) Eukaryotic Vector Of Neisseria Gonorrhoeae: Constuction And Immune Effect

One of the classic sexually transmitted diseases—gonorrhoeae is cause by the Neisseria gonorrhoeae. Human is the unique natural host, mainly spread by sexual intercourse. After infection the damage happened to the genitourinary system, cause to urethritis, cervicitis, and epididymitis, pelvic inflammation, etc. The gonorrhoeae has a damage on the physical and mental health, especially for the female with the complications in infertility, and the ectopic pregnancy, etc. which brings the severity result. The gonorrhea is popular in the globe and according to the WHO reports,there are about annually 62,000,000 new cases in the world. The infection is concerned with education level, sexual behavior method and the immunity, etc. and has a change in different region and season. In the United States, the gonorrhea incidence rate is higher in man,but the patience most are carrier in the women. The United States enforces the gonorrhea control plan, cause to the gonorrhea lowest level in the past 30 years, lower than the rate of nongonococcal urethritis. The situation of gonococcal infection in our country are still very seriously, according the monitoring in sixteen cities in our country, in 1987 gonorrhea sufferer was 11,115, there were 224,331 in 1997, in 1999 the number achieve to 340,960 example, the rate of increase in years was more than 30%, the infection growth rate was 20.63/100,000, stand head of the sexual transmitted disease. The guideline dropped in 2000, the sufferer was 285,661, in 2001 was 234,561, come second.The Neisseria gonorrhoeae is etiological agent of gonorrhea, mainly causing acute or chronic pyogenic infection on the genitourinary system mucosa. Recently researches discover that outer membrane protein has play a impotant role in the disease process and the host immune response, its outer membrane protein has at least three kinds, be respectively called outer membrane proteinⅠ(PⅠ), outer membrane proteinⅡ(PⅡ) and outer membrane proteinⅢ(the PⅢ). Among them the PⅠis the main out membrane protein, accounting for 60% of outer membrane protein above, forms porous channel on the cell membrane, and then called the Porin(Por) protein. The serotype mainly has the PⅠA and the PⅠB, made up of water-soluble material,have the material of the important function to the germ metabolism and some antibiotics to enter cell through the cell membrane. The PⅡcontributes to the adhere of Neisseria gonorrhoeae to mankind epithelioid cell, posses hot modificability, called Opa. The PⅢhas the deoxidize modificability, called Rmp. The outer membrane protein is a macromolecule material, can become the antigen to stimulate organism to produce corresponding antibody. Pathogenic mechanism of Neisseria gonorrhoeae is very complex,for attachment is the precondition of pathopoiesis. Neisseria gonorrhoeae needs to attach to mucosa of the genitourinary tract first after entering the organism, then causes a series of pathological affection. Previously thought pilus is the attachment structure, but recently research indicates that the outer membrane protein in the process also is importance. The PⅠis the main composition in the outside membrane, becomes more and more valuable because of its opposite and conservative gene sequence and stable expression, we already confirm that purified PⅠform the electric voltage dependence type ion choice passage in the membrane of eukaryotic cell, which contacts with the pathopoiesis. The contribution of the PⅡ(Opa) in the attachment to the host cell has already got generally accepted, already some scientist make use of the Western blot to confirm the PⅠespecially PⅠA can attach and infringe upon eukaryotic cell without PⅡ. The function of the PⅠA attachment to epithelial cell demands the hole to be placed in the open appearance, providing the electric voltage dependence binding site thus with/or imbed of cell surface enduce its function. The bulk of PⅠin the outer membrane protein is trimer is contacted with the PⅢforms compound. The PⅢis exposed along with the variety of the PⅠserum type on the surface, so we can doubt in the process of PⅠattachment whether the PⅢhas an actual function. VanPutten, etc. make use of the method knockout to knock the PⅢgene to confirm that without PⅢthe PⅠA is function as leading role. Owing to the structure characteristics of that germ and with the result that the disease characteristics, utilizing a complete sequence of the PⅠA to construct Eukaryotic vector(pCI-PⅠA) for gonorrhoea, we observed the expression of PⅠA encoded by the gene in cytoplasm and on cytomembrane of mice to study immune reaction of Balb/c mice and bacteria-killing function of the antibody cooperating with complement in serum.The studies were divided into two parts:Part one Eukaryotic vector (pCI-PIA) of Neisseria gonorrhoeae: Construction, appraisal and expression in vivo1,The Construction and appraisal eukaryotic vector (pCI-PⅠA) of PⅠAUsing PⅠA 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 pCI-neo were digested with restriction endonuclease EcoR I and Sal I, the larger fragments were reclaimed after 1% agarose electrophoresis. The larger fragments from pCI-neo and PⅠA 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 PⅠA and amino acid of Neisseria gonorrhoeae from the gene library. The DNA sequence demonstrated that the purpose DNA was agreeable to the one (AF090820) recorded in the GenBank, indicating that there was 99.12% identity for nucleotide. Seven bases were changed in property. It was expected that five amino acids underwent permutation in the primary structure of protein. One of them was shown a synonymous mutation, resulting in the transformations of amino acid as 24 Try→Cys, 87 Thr→Asn, 213 Gly→Ala, 247 Val→Leu, 257 Asn→His, others were synonymous mutation.2,The expression of eukaryotic vector (pCI-PⅠA) of Neisseria gonorrhoeae in vivoNine Balb/c mice were divided into three groups with three in each group, pCI-PⅠA,pCI-neo and physiological saline were injected to the animals of three groups, respectively, with the dose of 100ug/35ul for each animal. After ten days of the first immunization, the injections were repeated with the same doses to boost their immunizations. Four days after the final immunization, the mice were killed and paraffin sections was prepared with bilateral quadriceps muscle. Eukaryotic vector pCI- PⅠA in the expression of skeletal muscle cell of mice was examined with immunohistochemical method. It was found that Eukaryotic vector was presented in the membrane of skeletal muscle cell and the cytoplasm of the mice, which indicates the success of the development of Eukaryotic vector(pCI-PⅠA) of Neisseria gonorrhoeae. Part Two Studies on the immunity effect induced by Neisseria gonorrhoeae's DNA vaccine(pCI-PIA)1,The inoculation of Neisseria gonorrhoeae's eukaryotic vector pCI-PⅠAThirty Balb/c mice were randomly divided into 3 groups in average and vaccinated three times at 3-week intervals. At week 0 mice was immunized by intramuscular with Eukaryotic vector pCI-PⅠA(100ug/35ul for each),plasmid pCI-neo(100ug/35ul for each animal)and physiological saline(35ul for each animal)at posterior tibialis. At week 3 and week 6 mice were boosted with pCI-PⅠA and pCI-neo. At the week 0 (prior to immunization), 3, 6, 8, 10, 12, 16 and 18 weeks, 20ul of mice serum were collected from the vena caudalis. The serum was drawn at the week 22 from orbit of the mice. Before and after the immunizations given to the mice, the level of special antibody IgG in serum were analyzed by ELISA. The results showed that the titer of IgG in serum, gradually increased as the time of immunization was prolonged. At the week 10 it reached its maximum (1:4000), and maintained this level until the week 12, and then it started falling gradually. It dropped to its minimum at the week 18.2,Observation of bactericidal activity of eukaryotic vector pCI-PⅠAThe living Neisseria gonorrhoeae were added in immune serum. After incubation for 15 min at 37℃, fresh and normal mouse's serum(containing complement) were added to these liquid and incubation for another 30 min. The Neisseria gonorrhoeae mentioned above were put on Chocolate solid culture medium and then cultured for 24~48 h. After 24~48 hours, we checked the results. The control groups were also made for another reaction system(Ag-Ab complex) without an addition of complement. Physiological saline was used for negative control group. Immune serum can kill bacteria completely with the complement synergistic action(no bacteria were seen in culture). The bacteria grown fully on the culture medium in negative control group. The number of the bacteria on the control culture of Ag-Ab complex were much less than that in the negative culture's, but more than that on the culture of Ag-Ab complex with complement.3,T lymphproliferation response After seven days of the final immune of Eukaryotic vectors, executed mouse and got the spleen to prepare T lymphocyte cell suspension, regulated the cell density to 2×106 /ml, took 100ul cell suspension to every hole of shadow mask, then added purified PⅠA to experiment team and physiological saline to contrast team, incubate in CO2 thermostated container for 72 hours, add in the MTT, incubate for 4 hours, survey the OD570, the statistical analysis of experiment result which prove the strength of the specificity proliferation of T lymphocyte after immunity.Conclusions:There is no studies reported in China about immune protective effect of the major outer mambrane antigen(PⅠA) of Neisseria gonorrhoeae. In this study, we employed molecular biological technology to successfully construct the Eukaryotic vector of the major outer membrane protein of Neisseria gonorrhoeae pCI- PⅠA. Using Eukaryotic vector to immunize Balb/c mice, it was found that the mouse showed the signs of immune protection, and that immune serum exhibited capability to kill bacteria in vitro. Because protein vaccine needs to be purified, a tremendous amount of work is generally required for the mass production. Furthermore, protein vaccine needs to be repeatedly inoculated so as to achieve better immune results. DNA vaccine shows a great advantage over protein vaccine as a result of its procedures that is much simpler than recombinant protein vaccine to produce DNA vaccines. Consequently, the mass production of DNA vaccines can be made without huge investment. The quality control is easily implemented. Their low cost and stability to the temperature that incurs no need to store the vaccines in the refrigerator are appealing. All of these advantages demonstrate that the DNA vaccine will potentially become an effective nucleic acid vaccine(DNA vaccine).