Cloning,expression and identification of the different fragments of polymorphic membrane protein I and its immunogenicity analysis of Chlamydia trachomatis serovar D / 中华传染病杂志

Objective To obtain the full length (FL ) and C‐terminal fragment of polymorphic membrane protein I (PmpI) of Chlamydia trachomatis serovar D ,and to study the immunogenicity of these proteins .Methods The target genes of PmpI‐FL and PmpI‐C were amplified by polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid vector pGEX‐6P‐1 .The recombinant plasmids pGEX‐6P‐1/PmpI‐FL and pGEX‐6P‐1/PmpI‐C were separately transformed into Escherichia .coli ( E . coli) DH5αand were identified by enzyme digestion ,sequencing and PCR .After the identification ,the recombinant plasmids were separately transformed into E .coli BL21 and induced to express the proteins . The expected proteins were identified by Coomassie brilliant blue staining and Western blot ,then purified by glutathione S‐transferase (GST) MagBeads .The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI‐FL or PmpI‐C .Enzyme‐linked immune sorbent assay (ELISA) was used for the quantitative detection of the specific antibody .Results The lengths of cloned target genes PmpI‐FL and PmpI‐C were 2 659 bp and 1 195 bp ,respectively ,and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank .The molecular masses of target proteins were 122 000 and 69 000 ,respectively ,which were confirmed by Coomassie brilliant blue staining and Western blot and then purified .The titers of the antibodies (anti‐PmpI‐FL and anti‐PmpI‐C) in sera of immunized mice detected by ELISA were 1∶12 800 and 1∶6 400 ,respectively .Conclusion The PmpI‐FL‐GST and PmpI‐C‐GST fusion proteins with high immunogenicity are successfully expressed and purified , which lays the foundation for further study .

Immune responses induced by the recombinant major outer membrane protein vaccine against Chlamydia trachomatis E serotype in rhesus monkeys / 中华皮肤科杂志

[Objective] To observe the specific immune responses induced by the recombinant major outer membrane protein (rMOMP) vaccine against Chlamydia trachomatis E serotype in rhesus monkeys.[Methods] Six rhesus monkeys were equally divided into three groups:adjuvant and protein group vaccinated with purified rMOMP and Freund's adjuvants,adjuvant group immunized with Freund's adjuvants only,and control group immunized with phosphate buffer.All the rhesus monkeys were intramuscularly immunized in the triceps brachii for 3 times at a 2-week interval.Two weeks after the last vaccination,serum,vaginal wash and venous blood samples were collected from the rhesus monkeys,and lymphocytes were isolated from the blood samples.Enzyme linked immunosorbent assay (ELISA) was performed to determine the specific IgG antibody and interferon level in sera and secretory IgA (sIgA) level in wash samples,and methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferation of lymphocytes after stimulation with Chlaraydia trachomatis serotype E elementary bodies.Delayed hypersensitivity was observed in rhesus monkeys challenged by inactivated Chlamydia trachomatis serotype E elementary bodies.In vitro antibody neutralization assay was conducted with the serum from rhesus monkeys.Indirect immunofluorescenee was used to detect Chlamydia trachomatis in exfoliative vaginal cells from rhesus monkeys from week 1 to 10 after challenge with Chlamydia trachomatis.Data were statistically analyzed by using one-way analysis of variance and least significant difference (LSD) test with the SPSS 14.0 software.[Results] The adjuvant and protein group differed statistically from the adjuvant group and control group in the serum level of specific IgG antibody (1.718 ± 0.213 vs.0.841 ± 0.315 and 0.791 ±0.437,both P＜ 0.05),interferon ((1086 ± 121.730) ng/L vs.(409 + 53.440) ng/L and (162 ± 48.046) ng/L,both P＜ 0.05),lymphocyte proliferation index (7.012 ± 1.026 vs.4.473 ± 1.850 and 1A26 ± 1.104,both P＜0.01 ) and the diameter of nodus in delayed hypersensitivity assay ( ( 1 1 ± 2.134) mm vs.(3 ± 0.914) mm and 0,both P ＜ 0.01 ).After attack,the exfoliative cells kept positive for Chlamydia trachomatis in the adjuvant and protein group from week 1 to 5,and in the other 2 groups from week 1 to 10,but were negative in the adjuvant and protein group from week 6 to 10.[Conclusion] The rMOMP vaccine can induce a specific,protective,humoral and cellular immune response against Chlamydia tracbomatis in rhesus monkeys.

The cross immune responses of the rhesus monkey induced by the E style Chlamydia trachomatis recombinant major outer membrane protein / 中华微生物学和免疫学杂志

Objective To test cross immune responses induced in rhesus monkeys immunized with the recombinant major outer membrane protein(rMOMP).Methods Six rhesus monkeys were divided into three groups:the group vaccinated with purified rMOMP and Freund's adjutants,the group vaccinated with Freund's adjutants only and the control group vaccinated with PBS.All of the rhesus monkeys vaccinated intramuscularly at 0,2,4 weeks.Two weeks after the last time,The IFN-γand Chlamydia-specific antibody titers in sera,which were determined by ELISA,lymphocyte proliferation assay were performed by MTT,and observ the delayed hypersensitivity and in vitro neutralization assays.Results The result of the monkeys immunized with rMOMP and Freund's adjuvant:the specific immune responses can be observed.The in vitro neutralization and lymphocyte proliferation assays were observed better in the same group.Conclusion After being vaccinated with rMOMP,the monkeys can develop strong and effective Chlamydia-specific cross immune responses.

Preparation, identification and application of polyclonal antibody against major outer membrane protein of Chlamydia trachomatis E / 中华传染病杂志

Objective To express major outer membrane protein (MOMP) of Chlamydia trachomatis E and to prepare rabbit polyclonal antibody. Methods The recombinant plasmid MOMP/ pGEX6p-l prepared by our lab was introduced into E. coli. The protein was expressed and purified by gel recycling, then was injected into New Zealand rabbits to produce polyclonal antibodies. Enzyme linked immunosorbent assay (ELISA) was used to detect the titer of antibody. The antibody specificity was identified by Western blot and immunofluorescence. Results The fusion recombinant protein glutathione S-transferase (GST)-MOMP was successfully expressed in E. coli. The titer of antibody recombinant protein detected by Western blot and to the endogenous MOMP of Chlamydia trachomatis in vitro detected by immunofluorescence. Conclusions The recombinant MOMP is successfully expressed and the MOMP antibody with high titer and high specificity is obtained. which will be helpful for Chlamydia trachomatis detection and related clinical research.

Preparation and preliminary application of monoclonal antibody against Vp1 protein of chlamydiaphage ΦCPG1 / 中华皮肤科杂志

Objective To express recombinant Vp1 protein of chlamydiaphage φCPG1, prepare monoclonal antibody against Vp1 protein and utilize it to screen clinical isolates of Chlamydia trachomatis. Methods The Vp1 protein was obtained by prokaryotic expression, and monoclonal antibody against this protein was prepared by hybridoma technique. ELISA and Western blot were used to identify monoclonal antibodies. Then,the monoclonal antibody was prepared in quantity by injecting hybridoma cells into the abdominal cavity of BALB/C mice, and purified by using protein G affinity chromatography. Clinical isolates of Chlamydia trachomatis were screened for the chlamydiaphage by immumofluorescence assay using the prepared monoclonal antibody.Results Purified Vp1 protein was obtained. The monoclonal antibody against Vp1 protein was gained after 3times of sub-clone and consistently identified as IgG1. Three hybridoma cell strains that stably secreted monoclonal antibody were generated. Chromosome analysis of hybridoma cells showed that the mean number of chromosome was 96, most of them were telocentric and a few were submetacentric. The titer of purified monoclonal antibody was more than 1: 12 800. Twenty clinical isolates were screened by using the monoclonal antibody and no positive results were obtained. Conclusions The monoclonal antibody against Vp1 protein of chlamydiaphage φCPG1 is successfully prepared, while no chlamydiaphage is detected by immumofluorescence assay using the prepared antibody in 20 clinical isolates of Ct.

Detection of serum antibodies to polymorphic membrane proteins (Pmp) in patients with urogenital Chlamydia trachomatis infection / 中华皮肤科杂志

Objecfive To detect serum antibodies to Pmp in patients with urogenital Chlamydia trachomatis infection and to assess the relationship between Pmp and urogenital C.traehomatis infection.Methods Twenty healthy adults and 77 patients with urogenital C. trachomatis infection were recruited into this study.A 3-month foilow-up was carried out in 43 patients,who were classified into persistent infection group(n=19)and negative-conversion group(n=24).Western-blot was performed to detect serum antibodies to Pmp in all subjects.Results The positivity rate of anti-Pmp antibodies was 90.20% (71/77) in patients,significantly higher than that in the normal controls[20% (4/20),P<0.05].All the 9 types of anti-Pmp antibodies were detected in patients with a varying positivity rates,which were 61.04% (47/77),88.31% (68/77),63.63% (49/77),28.57% (22,77),63.63% (49/77),75.32% (58/77),62.34% (48/77),77.92% (60/77)and 70.13% (54/77) for antibodies against PmpA,PmpB,PmpC,PmpD,PmpE,PmpF,PmpG,PmpH and PmpI respectivelyThe prevalence was highest for anti-Pmp B antibodies and lowest for anti-Pmp D antibodies.There was no significant difference in the positivity rate of anti-Pmp antibodies between persistent infection group and negativeconversion group.Conclusions Anti-Pmp antibodies could be generated in patients infected with C. trachomatis.The immunogenicity of different Pmps is different,and the immunoprotective activity of Pmps is rather weak.Individual differences exist in serum anti-Pmp antibodies among patients.Nine types of Pmps are expressed in patients with urogenital C. trachomatis infection.

Cloning, expression, purification and identification of Chlamydia trachomatis polymorphic membrane protein G / 中华皮肤科杂志

Objective To clone, express and purify Chlamydia trachomatis polymorphic membrane protein (Pmp G), and to identify its immunogenicity. Methods The Pmp G gene of C. trachomatis serotype E was amplified by PCR, cloned into prokaryotic expression vector PET30a (+). The positive recombinant was transformed into the bacterium E coli (BL-21), identified by enzyme digestion, PCR amplification and gene sequencing. Then, it was induced to express followed by the identification of expression product with SDS-PAGE and Western blotting. The purified protein was used to immunize BALB/C mice to test its immunogenicity. Results PCR produced a 1092 bp-sized DNA fragment, which had a sequence consistent with that of PmpG gene of C. trachomatis E type in the GenBank database. The molecular weight of expression product was 55 kD, which was proved to be the expected size, and Western Blotting confirmed it to be the specific protein. Moreover, special antibodies to PmpG were induced to be generated by mice immunized with the purified protein. Conclusions The constructed prokaryotic expression vector for PmpG is expressed successfully in E. coli, and the expression product shows immunogenicity.

Construction of models for persistent genital tract infection with Chlamydia trachomatis in mice with low serum iron / 中华皮肤科杂志

Objective To construct a model for persistent genital tract infection with Chlamydia trachomatis in mice with low serum iron. Methods Iron deficiency was induced in female BALB/c mice by lowiron diet. Mice with or without iron-deficency were inoculated intravaginally with direct instillation of 50 μl of bacterial suspension containing 3.4344 × 107 IFU/ml of C. trachomatis serovar E elementary body 1 week after intramuscular injection of progesterone. Moxifloxacin and ferrous sulfate, alone or in combination were given to inoculated mice with iron deficiency daily for 1 week. Subsequently, vaginal discharge was obtained from mice and observed under an immunofluorescence microscope, and tissue specimens were resected from the uterus,uterine tube and ovary of mice and subjected to pathological examination at various time points. Results Compared with normal-iron mice, iron-deficient mice were infected with C. trachomatis for a longer duration (more than 18 weeks). After 18-week infection with C. trachomatis, there was an obvious chronic inflammation and lymphocytic infiltration in tissues specimens from the uterus,uterine tube and ovary of mice. Immunofluorescent examination of vaginal discharge for C. trachomatis turned negative in 60% of iron-deficient mice treated with both moxifloxacin and ferrous sulfate, but not in any of those receiving moxifloxacin or ferrous sulfate alone. Conclusion As animal models show, low serum iron may contribute to the persistent genital tract infection with C. trachomatis.

Immune responses induced by recombinant major outer membrane protein of Chlamydia trachomatis serovar E / 中华皮肤科杂志

Objective To investigate specific immune responses in mice induced by recombinant major outer membrane protein(rMOMP)of C.trachomatis serovaf E.Methods Thirty-six female BALB/cmice aged 3 to 4 weeks Were divided into three groups.i.e.,adjuvant group vaccinated、with purified rMOMP and Freund's adjutant,solitary group vaccinated with rMOMP only and control group vaccinated with phosphate buffered saline(PBS).All the mice were intramuscularly vaccinated on week 0,2 and 4.Blood samples and vaginal washes were obtained from these mice on week 6,then,mice were challenged with elementary body(EB)of C.trachomatis serovar E at the footpad followed by the observation of delayed hypersensitivity.On week 7.mice were genitally infected with C.trachomatis EB;one week later,blood samples and vaginal washes were obtained again;six weeks later,spleen lymphocytes were isolated from the mice and stimulated bv C.trachomatis or ConA followed by the detection of cell proliferation with MTT assay.In vitro neutralization assay was also performed.ELISA was used to determine the titers of Chlamydia-specific IgO antibody in sera and IgA antibody in vaginal washes,as well as the level of IFN-γ in culture supernatant of lymphocytes and sefa of mice.Vaginal swabs were collected after genital challenge and subjected to C.trachomatis culture.Results The absorbance at 405 ms of Chlamydia-specific IgG antibody and proliferation index of lymphocytes were 0.641±0.059 and 5.085±1.291.respectively,in mice immunized with rMOMP and Frennd's adjuvant.significantly higher than those in mice immunized with rMOMP only(0.424±0.015 and 3.123 ±0.840.both P<0.05).The thickness of right hind footpad increased by 0.324±0.054 mm and 0.272±0.064 mm,respectively,in solitary group and adjuvant group,respectively,with significant difference between the two groups(P<0.05).A significant increase was also observed in the adjuvant group compared with the control group in the above three parameters(all P<0.01).Conclusion The rMOMP of C.trachomatis could efficiently induce Chlamydia-specific humoml and cellular immune responses in mice.