Prokaryotic Expression And Immunogenicity Analysis Of Six Inc Proteins Of Chlamydial Pneumoniae

Based on epidemiological investigation of Cpn infection amongindividuals with pulmonary disease, cardiovascular and cerebrovasculardiseases and healthy individuals, immunogenicity of the six known Incproteins, Cpn0146, Cpn0147, Cpn0186, Cpn0308, Cpn0585, Cpn1027, wasfurther analysed in people affected naturally with Cpn. This will providethe basis for the following researches.According to the literature information, six genes coding Inc proteinswere selected and bioinformatics for the six proteins were analyzed withExpasy and antheprot2000 software. Six proteins'formula, Mooreextinction coefficient of solution, half-life stability coefficient, aliphaticindex, total hydrophilic sites and modification were acquired by Expasyanalysis; six proteins'antigenicity, hydrophobic, solubility, hydrophilic,transmembrane domain and the secondary structure were analyzed byAntheprot2000 software. Inc protein of C. pneumoniae has their peculiarhydrophobic transmembrane region, which locates in 40-100AA and ismade up of more than 60AA.88 pulmonary disease clinical samples, 62 cardiovascular andcerebrovascular disease clinical samples and 24 healthy individuals clinicalsamples were collected. CpnIgG,CpnIgM and CpnIgA were determinedby indirect ELISA and CPn DNA coding 16sRNA was detected by PCR.Among 62 cases of cerebrovascular disease, the positive rates of CpnIgG,CpnIgM, CpnIgA and CPnDNA were 67.7%, 50.0%, 61.3% and 71.6%respectively. CpnIgG, CpnIgA and CPn DNA 16SRNA in cerebrovasculardisease group had higher positive rate than those in control group(P<0.05).Among 88 cases of lung disease, the positive rates of CpnIgG, CpnIgM,CpnIgA and CpnDNA were 58.0%, 55.6%, 50.0%, 82.2% respectively. The positive rate of CPnDNA in lung disease group was significantlyhigher than that in control group(P<0.05). The positive rates of Cpn DNA,CPnIgA and CPnIgG in the patients with cardiovascular andcerebrovascular diseases were higher significantly than that of the controlgroup. In lung disease group, the positive rate of CpnDNAwas higher thanthat of control group.The six genes coding Inc proteins were amplified by PCR from theC.pneumoniae AR39 genome, and the PCR products were digested by therestriction enzymes BamHâ… and Notâ… , then ligated with pGEX-6P2vector treated with the same endoenzymes. The recombinant plasmids weretransformed into E.coil XL1-blue and positive clones were screenedthrough colony-PCR, Cross-PCR, sequencing and blasting. The GSTfusionproteins were induced by IPTG, then were purified by GlutathioneSepharose 4B with GST tagged to the N-terminus and identified withSDS-PAGE. The primers of six genes were designed and six recombinedgenes were obtained. Sequence analysis demonstrated that the homology ofsequence was 100% for all six cloned genes by blasting those sequenceswith gene bank.The positive colonies were selected and induced to expressthe recombined fusion proteins. After being purified, recombinant proteinswere identified by SDS-PAGE electrophoresis, the bands and molecularsize of those recombined fusion proteins were consistent with the expected.The antibody to Inc protein in the positive samples was measured withindirect ELISA respectively and confirmed by Western-blot. IndirectELISA results demonstrated that antibodies to CPn0147, CPn0308 andCPn0186 were still positive when plasma dilution was at 1:500. Thespecificity of Ab was confirmed by Western blotting. Of the six Incproteins, Cpn0147, CPn0308 and Cpn0186 have more strongimmunogencity in population infected with Cpn.