Construction And Expression Of The Major Outer Membrane Protein PI Of Neisseria Gonorrhoeae In Escherichia Coli

Object:Porin I (PI) is the major outer membrane protein of Neisseria gonorrhoeae. In this research we described a method by which the gene encoding PI was cloned into an expression plasmid and then PI protein was expressed in E.coil. The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of vaccines for prevention of Neisseria gonorrhoeae infection.Methods:1. Genome extraction of Neisseria gonorrhoeae. Four clinic isolates of Neisseria gonorrhoeae (named NG₁, NG₂, NG₃, and NG₄ respectively) were collected, and then the genome of these strains were extracted. The gene encoding for PI of Neisseria gonorrhoeae was amplified by PCR (polymerase chain reaction), with a pair of particular designed primers.2. Cloning and sequence analysis of Neisseria gonorrhoeae major outer membrane protein PI. The genes encoding for PI were inserted into the cloning vector pBS-T, transformated to E.coli DH5α competent cells. The subsequent clones were demonstrated as ampicillin resistance and white colonies. EcoR I and Xho I were applied to confirm the recombinant plasmids. And sequence analysis was also performed to verify the recombination plasmids and the diversity of the PI gene in four strains which infect Chinese.3. Construction of expression recombinant plasmids pET30b-PI and protein expression. PI gene fragments were purified and cut from pBS-T-PI recombinant plasmids, inserted into expression vector pET30b, transformated into E.coli DH5α competent cells. The target clones were selected by using Kanamycin resistance, and were detected by enzyme digestion and PCR. The conformed recombinant plasmids pET30b-PI was transformated to E.coli. BL21 (DE), and the the protein expression were induced by add IPTG in the inoculums. The expressed protein was analyzed bySDS-PAGE.Results:1. Four target outer membrane protein PI gene fragments designed as PI NGi x PI NG2x PI NG3 and PI NG4 were obtained by PCR from four clinic isolates of Neisseria gonorrhoeae (named NGi, NG2, NG3, and NG4 respectively). The lengths of these four fragments were range from 0.9kb to 1 .Okb.2. PI NGi > PI NG2> PI NG3 and PI NG4 were inserted separately into pBS-T vector to construct pBS-T-PI NGh pBS-T-PI NG2, pBS-T-PI NG3 and pBS-T-PI NG4 recombinants. These recombinants were conformed by both restrict enzyme digestion and sequencing.3. According to the sequencing data, the length of PI NGi and PI NG2 were 907bp and encoded a peptide of 302 amino acids. And the length of PI NG3 and PI NG4 were 970bp and encoded a peptide of 323 amino acids.4. In compare with the PI sequences in GenBank, PI NGi had two base differences with that of Neisseria gonorrhoeae 4805 strain (GI: 3925497//ACCESSION: AF090819). The homologous rate was 99%. PI NG2 had four base differences with that of Neisseria gonorrhoeae 4805 strain. The homologous rate was also 99%. PI NG3 had two base differences with that of Neisseria gonorrhoeae SU106 strain (GI: 1763329//ACCESSION: U75639) with the same 99% homologous rate. But the PI NG4 had a higher homologous rate (98%) with an 18 base differences when compared with that of 4174 strain (GI: 3925461//ACCESSION: AF090801). When compared within the four PI NGs, the homologous rate of the DNA was 74% with a 232 base differences, and the protein homologous rate was 67% accordingly. PI NGi and PI NG2 were more likely belong to PIA subtype whereas PI NG3 and PI NG4 were more likely sit in PIB subfamily.5. PI NGi x PI NG2> PI NG3 and PI NG4 were inserted separately into pET30b vector to construct pET30b-PI NGi, pET30b-PI NG2, pET30b-PI NG3 and pET30b-PI NG4 recombinants. These recombinants were conformed by both restrict enzyme digestion and PCR.6. pET30b-PI NGi, pET30b-PI NG2, pET30b-PI NG3 and pET30b-PI NG4 recombinants were transformed into BL21(DE3) expression host competent cells and protein expression were induced by adding IPTG. All strains but not pET30b-PI NG2 demonstrated obvious target protein bands on SDS-PAGE gel.Conclusion:1. Four target outer membrane protein PI gene fragments obtained from four clinic isolates of Neisseria gonorrhoeae were cloned successfully into pBS-T vector and four recombinant plasmids designed as pBS-T-PI NGi ^ pBS-T-PI NG2n pBS-T-PI NG3 and pBS-T-PI NG4 were obtained.2. Sequencing analysis on the recombinant pBS-T-PI NGs were made. This is the first sequence report on Neisseria gonorrhoeae outer membrane protein PI gene isolated from infected person in China. According to the DNA and protein sequences, PI NGi and PI NG2 were more likely belong to PI A subtype whereas PI NG3 and PI NG4 were more likely sit in PIB subfamily.3. The expression recombinant plasmids pET30b-PINGi, pET30b-PING2, pET30b-PING3, and pET30b-PING4 were constructed successfully. Target proteins were observed on SDS-PAGE gel. The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of preventve vaccines on Neisseria gonorrhoeae infection.