Expression And Purification Of Chlamydia Trachomatis Polymorphic Membrane Proteins And Studies On Its Clinical Detection

Chlamydia trachomatis (C.t) is the most common pathogen of sexuallytransmitted diseases in the world. Urogenital tract infection caused by D～Kserovars has different patterns of manifestation in clinic. Some patients havesubjective symptom, while some have no symptom; Some patients can be curedquickly, some are persisted and recurrent; some may result in severe complicationssuch as prostatitis, infertility, ectopic pregnancy and chronic pelvic inflammatorydisease. Other serovars of C.t can lead to trachoma and lymphogranulomavenereum. It has been an important problem of public health to prevent and controlC.t infection.Polymorphic membrane proteins (Pmps) are a new family of highly variableproteins found in outer membrane of Chlamydiae, which are unique to thechlamydiae. Props were presumed to have some cell wall associated function, such asadhesion, molecular transport, signaling, et al, and related with immune escape andtissue tropism. Pmps have been one of the new hot spots in the field of C.t study inthe world. Some research institutions of foreign countries are carring out the studyon relativity between Pmps and C.t urogenital tract infection.Objective: To clone, express and purify N terminal of Chlamydia trachomatispolymorphic membrane protein G(PmpG-N), and to identify its immunogenicity. Todetect Props' special antibodis in serum of patients who had urogenital tract infectiondue to C.t and discuss the relativity of Props with C.t urogenital tract infection.Methods: C.t E serotype Bour reference stain was cultured in McCoy cells. Thechromosome DNA was extracted as the template. PmpG-N gene was amplified by PCR and then cloned directionally into prokaryotic vector of PET30a(+) to form therecombinant plasmid. The recombinant plasmid was transformed into Ecoli DH5αtoexpress the plasmid. After being identified, the recombinant plasmid wastransformed into Ecoli BL21 to express PmpG-N. PmpG-N was purified by HisBandPurification Kit and its immunogenicity was identified with the method ofWestern-blot. Serum samples were collected from 77 C.t infected patients and 20health adults in clinic and Pmps special antibodies were detected by Western-blot.We compared the positive rate of Pmps-Ab in different groups and analyzed therelativity between the data with the clinical infection.Results: 1. PmpG-N of C.t E serotype Bour stain was cloned successfully andwell expressed in Ecoli BL21.2. Purified PmpG-N can conjugated with its specialantibody, which confirmed that it had immunogenicity. 3. The positive rate ofPmps-Ab was 90.20％in infected group and 20％in control group, the former wasmuch higher than the latter(p＜0.05). 4. Nine Pmps' special antibodies can bedetected in the serum of patients. The positive rates of different Pmps-Ab weredifferent, 61.04％, 88.31％, 63.63％, 28.57％, 63.63％, 75.32％, 62.34％, 77.92％and70.13％from PmpA to PmpI respectively; The positive rate of PmpB-Ab is thehighest, while that of PmpD-Ab was the lowest, much lower than that of PmpB-Ab(p＜0.05). 5. Comparing the positive rates of 9 Pmps-Ab in persistent infected groupwith that of the resolved group, there were no significant difference(p＞0.05). 6. Thepositive rate of PmpD-Ab in female group was 50％and 22.07％in male group, theformer was much higher than the latter (p＜0.05), there was no significant differencebetween the two group on the positive rate of other Pmp-Abs(p＞0.05). 7. Thepositive rate of 9 Pmp-Abs in youth group was much higher than that of middle andold age-group(p＜0.05).Conclusion: 1. PmpG-N of C.t E serotype Bour stain was cloned and expressedsuccessfully, it had immunogenicity. This work established the foundation for basaland clinical research on Pmps. 2. Pmps have immunogenicity, and the patiens of C.t urogenital tract infection can generate special antibodies to Pmps. 3. Theimmunogenicity of different Pmps is different, the immunogenicity of PmpB wasstronger, but PmpD's was weaker. 4. The immunoprotective function of Pmpswasn't strong. 5. Pmps' special antibodis can be detected in the serum of patients, butthere was individual difference and region difference. 6. There was also sexdifference and age difference.7. As nine Pmps' special antibodies can be detected inthe serum of patients, it can be confirmed indirectly that all of the nine Pmps of C.tcan be expressed.