Preliminary Study On Biological Characteristics Of Chlamydia Muridarum TC0668 Protein

Objective:The prokaryotic expression plasmid pGEX4T-tc0668 of the TC0668 protein of Chlamydia muridarum was constructed to induce and purify the GST-TC0668 fusion protein in E.coli.The target protein mouse polyclonal antibody and rabbit polyclonal antibody were prepared to determine the TC0668 in HeLa cells.Localization,phase expression,and induction of cytokine expression,laid the foundation for understanding the mechanism of TC0668 in the pathogenic process of Chlamydia muridarum,and then studying its cognate protein CT389 in Chlamydia trachomatis.Methods:1.The software was used to compare the similarity of TC0668 protein and Chlamydia trachomatis virulence factor CT389 protein.Using Sopma software to analyze its secondary structure.The on-line software Swiss-model was used to search for the protein with the closest structure to the TC0668 protein,and then the protein was used as a template to simulate the tertiary structure of the TC0668 protein.2.According to the sequence of tc0668 gene,specific PCR primers were designed,and tc0668 gene was amplified by PCR using Cm wild type genome as template.The reclaimed fragment was linked to the double enzyme cut pGEX4T-1,constructed the recombinant plasmid pGEX4T-tc0668,transformed the Escherichia coli?E.coli BL21?,induced the expression of the GST-TC0668 fusion protein by IPTG,and purified the GST-TC0668 fusion protein with the GST-agarose resin and then went to the endotoxin.3.The GST-TC0668 fusion protein was immunized with BALB/c mice and New Zealand rabbits respectively and the antiserum was collected.The reaction and titer of the antiserum and GST-TC0668 fusion protein were measured.The mouse derived polyclonal antibody and rabbit derived polyclonal antibody were purified to obtain the target protein.4.Single layer HeLa cells were prepared,and the laboratory preserved Cm Nigg II strains infected single layer HeLa cells of 0,4,8,12,16,20,and 24 h.The total RNA of the infected specimens at each time point after infection was collected,and the level of tc0668 was detected by real-time fluorescent quantitative PCR?Real-time quantitative PCR,RTq-PCR?.Indirect immunofluorescence assay?IFA?was used to detect the expression of TC0668 protein in 4,8,12,16,20 and 24 h infected specimens.5.Single layer HeLa cells were prepared,and the single layer HeLa cell 24 h was infected with Cm Nigg II strain.IncA,HSP60 and other proteins were used as control.The localization of TC0668 in infected cells infected with 24 h after 24 h was detected by IFA.It was further confirmed by IFA at different time points of infection cycle.6.Single-layer HeLa cells were prepared and monolayer cells were infected with Cm tc0668 single gene mutant strain(Cm tc0668^mut)and corresponding Cm tc0668 gene wild-type strain(Cm tc0668^wt),and supernatants and cells were collected 24 h later.Lysates were used to detect cytokine levels in cell supernatants and lysates using a protein chip.Results:1.Bioinformatics results showed that the similarity of tc0668 and ct389 genes and their encoded products was as high as 84%and 92%.The content of TC0668 protein?-helices,random coils and?sheets was17.4%,60.78%and 21.81%,respectively.2.The target fragment with a size of about 1227 bp was amplified by PCR,which was consistent with the expected size of tc0668 gene.The recombinant plasmid pGEX4T-tc0668 was connected to the expression vector pGEX4T-1.The recombinant plasmid was transformed into E.coli BL21 and IPTG to induce the expression of GST-TC0668 fusion protein with a molecular mass of about 72 kDa,which was consistent with the expected value.The GST-TC0668 fusion protein was purified by GST agarose resin column chromatography.3.The GST-TC0668 fusion protein immunized BALB/c mice and New Zealand rabbits respectively.The harvested immunized serum was purified to obtain mouse derived and rabbit derived polyclonal antibodies.The detection of polyclonal antibodies could identify the GST-TC0668 fusion protein,and the titer was more than 1:64000.4.IFA localize the endogenous protein expressed in HeLa cell 24 h infected by Cm Nigg II strain.The TC0668 antibody marker is similar to the IncA antibody marker,but is different from Pgp3 and HSP60.5.RTq-PCR results showed that after Cm infection of HeLa cells 4h,the tc0668 gene began to be transcribed,the expression of 16 h increased significantly and then maintained high transcriptional level.The results of WB and IFA showed that the TC0668 protein began to occur in 4h and increased significantly in 16 h,and continued to the end of the infection cycle.6.Supernatants and cell lysates were collected from 24 h cells infected with Cm tc0668^mutand Cm tc0668^wt,and cytokine expression was detected by protein chip.Compared with the Cm tc0668 gene wild-type strain,73 cytokines in the infected cell supernatant of tc0668 single gene mutant strain were down regulated,of which 36 were statistically different?P<0.05?,and 33 cytokines were up-regulated,among which 10 were statistically different?P<0.05?.In the collected cell lysates,compared to the Cm tc0668 gene wild-type strain,the up-regulated and down regulated cytokines in the infected cell lysate of the tc0668 single gene mutant strain were the same as those in the supernatant,and the other part was just the opposite.Conclusion:1.TC0668 protein may be an early secretory inclusion body membrane protein of Chlamydia,and TC0668 may enter the host cell nucleus through a certain pathway.2.TC0668 protein may affect the reproductive tract disease by regulating the expression of cytokines.