Cloning And Expression Of Polymorphic Membrane Protein D From Chlamydia Trachomatis And Studying Immunoprotection Of RCt-PmpD

Chlamydia trachomatis (Ct), which consists of 19 different serotypes, can cause human being many diseases. Ocular infections lead to trachoma, a chronic follicular conjunctivitis that results in scarring and blindness. The World Health Organization estimates that about 500 million people are suffered from trachoma which has lead 9 million patients to blindness, making Ct infection become the main factor of blindness. Urogenital infections, like urethritis, cervicitis, salpingitis et al., which caused by Ct, are the leading cause of sexually transmitted disease (STD) in industrialized nations. Ct infection has become one of the most serious problems to human being health, so controlling Ct infection needs effective vaccine.Surface-exposed antigens on the infectious elementary body (EB) are targets of protective antibody. The most extensively studied chlamydia Ag is the immunodominant antigen major outer membrane protein (MOMP), which, to date, is the known target of neutralizing antibody. Antibodies specific to MOMP neutralize the in vitro infectivity of Ct by blocking EB attachment. Vaccination of rodents with MOMP proteins or passive transfer of MOMP specific monoclonal antibody provides only partial immunity. However, MOMP might not be the most compelling vaccine candidate for use in humans because protection has been shown to be short-lived. Therefore, alternative targets of neutralizing Ab may be required to develop an efficacious human vaccine.Polymorphic membrane protein D (PmpD) is surface-exposed, with a mass of 155-kD, can be recognized by convalescent sera from individuals with lymphogranuloma venereum (LGV), cervicitis, urethritis and trachoma.Neutralization test was performed in hamster kidney (HaK) cells, PmpD antiserum can neutralize all Ct serotypes, suggesting Ct PmpD is a pan-neutralizing target, and, in theoretically, would provide protection against all human strains.In the study, primers were designed according to Ct PmpD gene sequence which was searched on the web site of NCBI, and the gene of PmpD was amplified from the purified genomic DNA of Ct L2 by PCR. 1% agarose gel electrophoresis analysis showed that the amplified product was approximately 2250 bp, coincided with the reported length. The amplified product and pET32a vector were digested with Nco I and Sal I respectively, then the expression plasmid pET32a-PmpD was then constructed by T4 ligase. After that, the recombinant plasmid was transferred into the host cells E.coli BL21(DE3) to obtain an engineered expression strain. By sequencing and identification, the open reading frame was correct, and the sequence identity was 99%, with one nucleotide mutation at site 1936, from G to A. Due to thioredoxin, the weight of recombinant Ct PmpD (rCt-PmpD) was about 116kD. The expression of rCt-PmpD was optimized, under induction of IPTG at 1mM, 5hr incubated at 37℃rCt-PmpD was highly expressed with inclusion body in E.coli.After ultrasonic homogenation and cleaning, the purified inclusion was acquired, and the purity of rCt-PmpD was over 85%. The purified rCt-PmpD was solved with 8mol/L urea, then renatured in the solution of 100mmol/L Tris-HCl, 2mmol/L EDTA pH8.0, 0.2mol/L Arginine, 2mol/L Urea, 0.004mol/L oxidized glutathione and 0.002mol/L reduced glutathione. After 24hr at 4℃, the solution was changed to distilled water. The antigenicity of rCt-PmpD was analyzed by WB with Ct L2 rabbit serum, the result showed that rCt-PmpD displayed good antigenicity.The antiserum was prepared after immuning the rabbits. The immunoreactivity of rCt-PmpD antiserum was detected by indirect immunofluorescence, the normal serum and LPS monoclonal antibody were used as negative and positive control respectively. The results revealed anti-rCt-PmpD antibodies recognized Ct L2 specifically. The Ct L2 was incubated with rCt-PmpD antiserum in vitro, then inoculated to the SPF eggs. The neutralization test indicated that the eggs were effectively protected by anti-rCt-PmpD rabbit serum.These findings showed that the recombinant expression vector (pET32a-PmpD) was successfully constructed. The recombinant protein exhibited a good antigenicity, and the anti-rCt-PmpD serum can neutralize the Ct L2 infection. These results may provide the foundation for further development on PmpD subunit vaccine.