Clone And Expression Of Neisseria Meningitidis Surface Protein A Gene

Neisseria meningitidis causes epidemic meningitidis. The incidence per year of meningococcal diseases during endemic periods is normally 1-3 cases per 100,000, butit can be as high as 500 per 100,000 during epidemics. This pathogenic bacteria primarily affects young children between 6 month and 2 year of age, but often infects teenagers. Recently, in our country, the incidence of epidemic meningitis had a trend of markedly increasing and pandemic disease could always be identified which severely threatened the health of our people.Neisseria meningitidis is classified into 12 serogroups based on the immuno-logical characteristics of the capsular polysaccharidesfound at their surface. Approximately 90% of all meningococcal diseases worldwide are caused by isolates of serogroups A, B, and C. Vaccines based on the capsular polysaccharides of serogroups A, C were developed and proved efficient to control outbreaks and epidemics of meningococcal diseases. However, these vaccines are poorly immuno-genic in very young children. Moreover, they do not induce immunological memory and the duration of the protection they provide is relatively short. Attempts to develop an efficient vaccine against serogroup B isolates were unsuccessful because the capsular polysaccharides of serogroups B might discourage attempts to improve the immunogenicity . For these reason, it is believed that non - capsular surface antigens shown to stimulate bactericidal antibodies should be considered as the prime vaccines candidates.Within serogroups, different serotypes and subtypes can be identified based on the antigenic specificity of the major outer membrane proteins ( OMP) . Forexample, the Group B has more than 20 serotype and each serotype has more than 10 subserotype. Subsequently, there are more than one thousand surface proteins of the twelve serogroups. To search for the mutual surface proteins of all groups of Neisseria meningitidis had been the objective of all the researchers from everywhere of this world in recent several decades.A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified in 1997 by Martin . Nucleotide sequencing revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues. Although the function of NspA is not so clear, but the injection of purified recombinant NspA protein efficiently protect mice against a bacterial challenge with a lethal dose of Neisseria meningitidis .NspA would be significantly valuable to diagnosis and prevention of the epidemic meningitis. Therefore,we cloned NspA gene and expressed NspA fusion protein . We made ELISA examination with the sera from 10 patients who were in their recovery stage, the result showed that all patients in their recovery possessed the anti — NspA antibody .Materials and MethodsNeisseria meningitidis cells were scraped from agar and resuspended in 400 |xl ddH2O. bacterial suspension in water were boiled at 100T! for 10 minutes and centrifuged at 13000 g for 10 minutes, the supernatant was used as template per PCR . Amplification of the DNA was achieved by using the enzyme Pyrobest, a 50 - jxl reaction mixture contained 34 jxl ddH2O, 1 fxl of each primer, 5 fjd of 10 x buffer, 4 jxl of deoxynucleoside triphosphates , 1 jxl of Pyrobest enzyme, and 4 jxl of template DNA. Amplification was performed by using 30 cycles of denat-uration at 94 °C for 1 min , annealing at 60 °C for 1 min, and extension at 72 C for 2 min. the amplified product and the pGEX -5X -3 plasmid were digested with BamHI and EcoRI restriction endonucleases . The DNA fragments were separated by electrophoresis, and were recovered from agarose and purified by using the DNA purification kit . The NspA DNA and pGEX - 5X - 3 vectorswere ligated with T4 DNA ligase and transformed into E. coli BL21. Transfor-mants were analyzed by restriction endonuclease digestion of the resultant plas-mid ( pGEX - 5X - 3 ( + ) NspA) and sequencing of the coding region of the Ns-pA gene.An overnight transformants culture (1 % ) was used to inoculate one flasks containing 100 ml of LB media . The flasks were incubated at 30 C with vigorous shaking (225 rpm) until an OD^ of 0. 6 was reached. To induce protein expression , IPTG was added to a final concentration of 250 mmol/L. the cultures were incubated for 2 h . The body was harvested by centrifuged at 6,000 g for 10 min at 4 C. GST - NspA was purified by GSTrapFF affinity chromatography. Its purity was analyzed by SDS - PAGE and its concentration was detected by Bradford method.Enzyme - linked immunosorbent assay ( ELISA) procedure: A volume of lOOul of recombinant NspA protein diluted in 50mmol/L carbonate buffer at a concentration of 4. Oug of protein per ml was dispensed in each well of microti-terplate and adsorbed for 18 h at room temperature. The microtiterplate was washed with PBS containing 0.02% Tween 20 , and 200ul of 0. 1% (wt/vol) bovine serum albumin was added to each well. After 30 min at 37 C, the BSA was discarded and the microtiterplate was washed, patients sera were added to each well, and the plate was incubated for 1 h at 37°C. A normal serum was used as negative control. After 1 h incubation at 37°C, the plate was washed, and lOOjxl of OPD was added. 15 minutes later the absorbance of the solution was determined at reading of 492 nm OD.ResultsUsing the DNA of Group B Neisseria meningitidis as the template, and after the PCR amplifying the visible band was around the 500bp marker and accorded with the actual value. We examined pFEX -5X-3( +) NspA plasmids using BamHI and EcoRI double - digesting and later, agarose electrophoresis showed bands accorded with the actual value. The NspA gene sequencing verified the total length of the NspA gene was 525 bp which encoded 174 amino acids andcompared with the correlated sequence in Genbank there was no novel mutant point. For identifying optimal expression condition, after adding IPTG we checked the bacterial culture media at 1 hour, 1. 5 hour, 2 hour and 2. 5 hour and finally made sure that the optimal expression time was 2 hour. NspA fusion protein was purified by GSTrapFF and SDS - PAGE showed its moleculer weight was 44KD.Examining 10 patients'sera who were in their recovery stage by ELISA, all patients in recovery stage showed anti - NspA antibody activity and their titers all above 1:20 which certified that the researcher expressed the bioactive NspA fusion protein.DiscussionFrom 1997 when NspA gene was firstly discovered, many researchers had studied it. Some researchers inoculated the mice with purified NspA and after 9 weeks, attacked the mice with lethal dose of the Neisseria meningitidis, the result was that within 72 hours 80% of all mice had survived and in the subsequent 2 months no death had been observed, which all proved that NspA - induced antibody was the protective antibody. However, until in recent, all experimental data were obtained from the animal and not a single researcher proved that NspA could induce protective antibody when injected into human body.We cloned NspA gene and successfully expressed the fusion protein of GST and NspA . We also made the ELISA examination with the sera from 10 patients who were in their recovery stage, the result showed that all patients in their recovery possessed the anti - NspA antibody and titers were all above 1:20, this not only had proven that NspA fusion protein possessed the bioactivity, and even more, this had proven when under natural infectious course, human being could generate antibody directly against NspA and this had paved the way leading to production of NspA as the subunit protein vaccine.Furthermore, the ELISA examining kit which was coated with NspA fusion protein could be utilized as a dynamic monitor of the human antibody levels, and acquired the significance of effective strategy for the prophylaxis of epidemicmeningitis.ConclusionWe successfully cloned NspA gene and express the NspA fusion protein . Our study first proved when under natural infectious condition, human being could generate antibody directly against NspA and this had paved the way for production of NspA as the subunit protein vaccine.