Identification And Characterization Of The Chlamydia Pneumoniae Hypothetical Protein CPn0308

Chlamydia pneumoniae are obligate intracellular parasites of eukaryotic cell with a unique biphasic life cycle. Since chlamydial organisms accomplish all their biosynthesis and replication within the cytoplasmic vacuole (designated as inclusion) of the infected cell. In order to maintain a successful intra- vacuolar growth, Chlamydia has to exchange both materials and signals with host cells via the inclusion membrane. Since Rockey et al. identified the first Inc (IncA) in 1995, more Incs have been found and more information on Inc function has been obtained. The studies have demonstrated that Inc proteins can actively interact with the host cells, potentially benefiting C. pneumoniae growth and contributing to C. pneumoniae pathogenesis.Searching for novel Inc proteins may help unravel the molecular basis of chlamydial interactions with host cells and has thus become a hot topic in the chlamydial research field. Computer program, which based on the hydro- phobic feature of identified Inc proteins, predicted 90 CPn candidate Inc proteins by autoscreeing open reading frame of Chlamydia pneumonia, but only a few were proved to be in inclusion membrane by antiboby labeling method. Evidences suggest that not all predicted Inc proteins by the computer prediction are localized in the inclusion membrane of chlamydial organism- infected cells and not all Inc proteins can be predicted by this approach. Therefore, there is an urgent need to directly identify/confirm new Inc proteins in the C. pneumoniae-infected cells using chlamydial protein-specific reagents and to further study their biology functions, and it will benefit to deeply understand the Chlamydial biology, pathogenesis and prevention.CPn0308 is one of the predicted Inc proteins,it needs further to experi- mentally confirm whether CPn0308 indeed is localized in the inclusion membrane within infected host cell. Hence, this study is to analysis charcaterics of CPn0308 and its associated genes through CPn gene bank, to clone CPn0308 and its related genes and express them by GST fusion protein model, to make polyclonal antibodies and monoclonal antibodies to these GST fusion proteins, to localize the endogenous proteins by antibody labeling method, and to further characterize the endogenous CPn0308 protein. It will provide importmant information on the potential roles of Inc proteins in C. pneumoniae pathogenesis and prevention.Part one Cloning and expression of CPn0308 and its related genes Objective: To analysis structure feature of CPn0308 and its associated genes with the help of computer prediction, including CT249, MoPn0520 and GPIC0474, and to further clone these genes and express GST fusion proteins, which will provide the basis for the following antibodies preparation and Inc protein identification.Methods:①The information of CPn0308 was retrieved according to reference mentioned. On Basis of the retrieved hints, the relationship among them was further analyzed.②The primers were designed to clone the genes, including CPn0308, CT249, MoPn0520, GPIC0474 and to screen pGEX-6P2. The target genes were obtained by PCR.③The target genes amplified were purified by phenol-chloroform method and further digested by restriction enzymes.④After being purified, the digested PCR products were linked with pre-treated pGEX-6P2 vector via T4 ligase, the recombinants were trans- formed into competent cell(XL1-blue), then cultured on the selected LB plates contained 100μg/ml AMP overnight at 37℃.⑤The positive colonies were selected using PCR directed towards pGEX-6P2 vector, the plasmids were extracted from the positive colonies and further identified using cross-PCR, the positive plasmids were sequenced and analyzed.⑥After the cross-PCR and sequence analysis, the correct colonies were induced with IPTG and the GST fusion proteins were expressed and purified by Glutathione Sepharose TM 4B, then identified with SDS-PAGE gel electrophoresis.Results:①CPn0308 gene with 366bps in length encoded 121 amino acid residues with the molecular weight of 12.971kDa was cloned. CT249, MoPn- 0520 and GPIC0474 which were similar to the CPn0308 in gene structure were also cloned. A gene consisted one cluster alone, was flanked by two known genes, glgP (alpha glycan phosphorylase) and dnaA, the number of the amino acid residues which was encoded by its corresponding gene respectively was similar.②After being digested by the BamHI and NotI, the recombinant pGEX-6P2/CPn0308, pGEX-6P2/CT249, pGEX-6P2/MoPn0520 and pGEX-6P2/GPIC0474 were obtained via directional insertion those digested products into pGEX6P2 vector pre-treated with the same way as that of the purified PCR products. After initial screening of recombinants using primers designed against pGEX6P2, recombinant plasmids were extracted from the positive colonies and confirmed by cross-PCR using one primer from pGEX6P2 and another from target gene. The amplified products were same as the expected ones respectively. The nucleotide sequence of the recombinant plasmid pGEX-6P2/CPn0308, pGEX-6P2/CT249, pGEX-6P2/ MoPn0520 and pGEX-6P2/GPIC0474 was analyzed by DNA sequence analyzer. By blasting those sequences with gene bank, the homology of sequence was 100% for all four cloned genes. All of them possess two transmembrane regions.③The selected positive colonies were induced to express recombinant protein. After being purified, recombinant proteins were identified by SDS-PAGE electrophoresis, the bands and molecular size of those recombined fusion proteins were consistent with the expected ones respectively.Conclusion:①The gene information on CPn0308 and its related genes, including CT249,MoPn0520 and GPIC0474, has been analyzed and they share a similar gene structure.②T he target genes, including CPn0308, CT249, MoPn0520 and GPIC0474, were successfully cloned into pGEX-6P2 vector. The fusion proteins with the glutathione-s-transferase (GST) of CPn0308 and CT249, MoPn0520 and GPIC0474 have been expressed.Part two Preparation of antibodies against four GST fusion proteins and preliminarily applicationObjective: To make polyclonal antibodies(pAbs) to GST-CPn0308 and other three GST fusion proteins and monoclonal antibodies(mAbs) against GST-CPn0308. To localize the four endogenous proteins preliminarily using antibody-labeling method, and to lays the foundation for thoroughly inquiring the biology function of these hypothetical proteins.Methods:①The immunogens were made by purifying GST-CPn0308, GST-CT249, GST-MoPn0520 and GST-GPIC0474 with Glutathione Sepha- rose TM 4B. The purified fusion proteins were used to immunize mice as routine way, the pAbs to four fusion proteins were obtained by separating serum from whole blood. Besides above mentioned, mAb to GST-CPn0308 were also made by the household method.②To screen positive clones and titrate mAbs using indirect immunofluorescence assay (IFA).③The class and subclass of mAbs to CPn0308 were also identified by IFA. The recognition specificity and sites of mAbs were identified with Western Blotting method.④The mouse anti-fusion protein antibodies were used to preliminarily localize the four endogenous proteins in corresponding Chlamydia-infected cells using an IFA. In order to observe the homology between CPn0308 and other three proteins, apart from absorption experiment, antibody to GST-CPn0308 was also used to directly test HeLa cells infected with four different species Chlamydia using IFA.Results:①The sera were titred by IFA as following: CPn0308 was 1:2000, and that of MoPn0520, CT249 and GPIC0474 was 1:200, 1:2000 and 1:200 respectively.②The four hybridoma stains which produced mAbs to CPn0308 were obtained, designed as 2D7,3A6,3H5,5E10. All of them were IgG class. Of all, 2D7 was IgG3, 3A6 was IgG1, both 3H5 and 5E10 were IgG2b. All of them recognized N-terminus of CPn 0308.③IFA indicated that hypothetical protein CPn0308, CT249 and MoPn0520 were in the inclusion membrane,but hypothetical protein GPIC0474 was not localized in inclusion membrane.④Comparing with contrast, no inclusion membrane staining was found if anti-GST-CPn0308 antibodies were pre-absorbed with GST-CPn0308, but inclusion membrane staining could be observed when anti-GST-CPn0308 antibodies were pre-absorbed with GST-CT249, GST-MoPn0520 or GST- GPIC0474. Serum to GST-CPn0308 only recognized inclusion membrane within HeLa cells infected by AR39, but not recognized inclusion membrane within HeLa cells infected by other three species of Chlamydia.Conclusion:①The four hybridomas(2D7, 3A6, 3H5 and 5E10) have been successfully obtained. All are IgG class and recognize N-terminus of CPn0308.②Hypothetical proteins CPn0308, CT249 and MoPn0520, not GPIC0474 have been found to be localized in inclusion membrane with antibody-labeling method.③It has been proved experimentally that CPn0308 has no homologues with CT249, MoPn0520 or GPIC0474. Part three Preliminary study on characteristics of hypothetical protein CPn0308Objectives: To identify hypothetical protein CPn0308 using antibody labeling approach, and further to study its characterization preliminarily.Methods:①Using pAb or mAb to CPn0308 as first antibody, IFA was carried out to observe the localization of the hypothetical protein CPn0308 within infected cells under the fluorescence microscope.②Costaining of CPn0308 with four other proteins from chlamydia pneumonia (CPAFcp,IncA,MOMP and HSP60 as controls ) was processed to observe coloca- lization of CPn0308 with control proteins under the laser confocal microscope.③The recombinants pDsRed-C1/CPn0308 and pDsRed-C1/CPn0186 were transfected into HeLa cells using the lipofectamine 2000 transfection reagent to characterize the staining of the anti-CPn0308 antibodies under the fluorescence microscope. Absorption experiment was done to visualize specificity of the inclusion membrane staining using antibodies to CPn0308 or IncA. The Western Blotting assay was carried out for testing specificity of the anti-CPn0308 antibodies reacted with endogenous and exogenous CPn0308.④The HeLa cells were infected with AR39 organisms for various periods of time, and the culture samples were subjected to IFA to test the expression model of endogenous CPn0308.Results:①The hypothetical protein encoded by C. pneumoniae open reading frame (ORF) of CPn0308 was detected preliminarily in inclusion membrane of C. pneumoniae-infected cells using antibodies raised with CPn0308 fusion proteins.②The anti-CPn0308 mAbs detected a dominant inclusion membrane signal, which was similar to the signal revealed by the anti-IncA, but not the anti-CPAF, anti-MOMP, or anti-HSP60 antibodies. The inclusion membrane localization of CPn0308 was further verified using the laser confocal microscopy. The anti-CPn0308 labeling did not colocalize with CPAFcp, MOMP, or HSP60 (staining showed green, red and blue color), but clearly overlapped with the anti-IncA labeling(staining demonstrated green and yellow color), even at different focal points along the Z axis.③The anti-CPn0308 antibodies only detected the RFP-CPn0308(yellow color)but not the RFP-IncA fusion proteins(red color). The detection of the endogenous antigens in the C. pneumoniae-infected cells by the anti-CPn0308 and anti- IncA antibodies was blocked by the corresponding homologous but not the heterologous GST fusion proteins in an immunofluorescence assay. In the Western Blotting assay, the anti-CPn0308 antibody only recognized a protein band corresponding to the endogenous CPn0308 from the lysates of C. pneumoniae-infected HeLa cells but not HeLa cell alone or C. trachomatis- infected cells.④When the expression of CPn0308 protein was monitored along the infection time, the CPn0308 antigen was detectable 24 hours after infection and remained in the inclusion membrane throughout the infection course.Conclusion:①The first experimental evidence has been provided to directly demonstrate the localization of CPn0308 in the C. pneumoniae inclusion membrane using the anti-CPn0308 pAbs and mAbs.②Coloca -lization demonstrates that distribution pattern of CPn0308 in inclusion membrane is similar to IncA, but not CPAFcp, MOMP and HSP60.③Time course of CPn0308 protein expression has been detected and it has been found that the CPn0308 protein was expressed at 24 h after infection and remained in the inclusion membrane throughout the infection course.Part four Analysis of CPn0308 immunogenicity in the infected patient with CPnObjectives:To analyze CPn0308 immunogenicity in the CPn infected patient with ICVD.Methods:①To collect clinical blood samples and separate plasma from blood and extract the DNA from PBMC.②The CPn IgG and CPn IgM were detected using CPn specified diagnose kit.③CPn DNA from PBMC was assayed using PCR.④Imunogenicity of the endogenous CPn0308 was analyzed by Western Blotting.Results:①Among the 70 patients with ICVD, the positive rate of CPn IgG was 51.43%, and it was higher than that of control; The positive rate of CPn IgM was 2.86%, and that of control was 8.47%.②The positive rate of CPn DNA in PBMC from patients with ICVD was 81.08%,and it was higher than that of control.③After plasma samples were diluted as 1:500, the antibodies to endogenous CPn0308 were still detected using Western Blotting. The reaction was blocked after the plasma was pre-absorbed with GST-CPn0308 beads ahead, but not pre-absorbed with GST-CPn0186 beads.Conclusion:①there is an increase in the positive rate of CPn IgG in the patients with ICVD.②The positive rate of CPn DNA in PBMC from the patients with ICVD is higher than that of the control. The positive rate for CPn by PCR is higher than that of EIA, and CPn DNA detection is a more sensitive method.③The studies preliminarily demonstrate that CPn0308 has immuno- genicity in the individual infected with CPn.