Recombinant Construction Of OmpA Gene From A Chlamydia Trachomatis Wild Strain Of J Sreotype

Chlamydia is an obligate , intracellular, Gram negative bacterial pathogen. The diseases evoked by chlamydia are quite wide. In developed countries, the pathogen is the leading cause of sexually transmitted disense. Infection in the eyes results in trachoma which once spreaded widely in the whole world. By now trachoma is still the leading cause of preventable blindness in the world. Chlamydia infection can also make cattle and sheep abort, hens not lay eggs normally. So chlamydia has made huge damage to both mankind and anminals. Chlamydia has became the important pathogen which absorbs people's attention. Chlamydial vaccion has the probability of controling chlamydial infection and spread. People have studied chlamydia vaccine for more then 50 years. Many kinds of vaccine have been tested, such as the inactivated pathogen vaccine, the live attenuated pathogen vaccine, the subunit vaccine, the dendritic cell vaccine and so on. But there isa't a mature vaccine that can be used now. In recent years, the fast and deep research OB the DNA vaccine brings new hope for stydying the chlamydia DNA vaccine.The DNA vaccine, which is a recombinant of plasmid with a gene fagment encoding antigen protein, vaccinate the organism directly , transform the organism cells, express the protective antigen, and then induce the organism to obtain specefic immunity.Our test is aming at constructing an recombinant of ompA gene from the chlamydia trachomatis wild strain. We choose pcDNA3 plasmid as the vector and ompA gene which encodes chlamydia trachomatis (C.t) major outer membrane protein as the target DNA. Clinical chlamydia trachomatis samples,urethral and cervical secreations, were collected from persons who presented to the outpatient department of the sexually transmitted disease. After lysised, clinical samples wert used as template for polymerase chain reaction(PCR). Firstly ,to select C.t positivesamples, we amplified ompA gene through PCR using C.t species specific primier, and then we ascertain the C.t clinical specimen's serotype by the DNA sequence method. Secondly, through polymerase chain reaction, we amplified specific serotype C.t ompA gene using serotype specific primier. Then we enzymatically inserted DNA fragment into plasmid vector, we used three methods to screen positive recombinant, including culturing the E.coli on LB agarose plate containing ampicillin, amplifing ompA gene from recomibant through the PCR method, and digesting recombinant by restriction endonuclease. Finally, we sequenced the inserted DNA fragment by sending the positive recombinant to the sequence corporation.The results are as follows. By using the PCR method to screen positive C.t clinical samples, the obtained DNA fragment is about 1.2Kb long. The sample which was sended to be sequenced was proved to be C.t J serotype. Agarose gel electrophoresis manifests that the amplified C.t J serotype ompA gene is a little longer than 1.0Kb. The length of the amplified DNA fragment is consistent whih what we have anticipated. When positive recombinant is screened by the PCR method, agarose gel electrophoresis manifests that ompA gene lies in pcDNA3 plasmid. Digested recombinant with restriction endonuclease, we obtained two DNA fragments of separately 5.4Kb and 1.0Kb. The result also confirms that the ompA gene lies in pcDNA3 plasmid. When we sequenced the inserted DNA fragment, the result proved that the inserted gene has the same sequence with C.t J serotype ompA gene provided by genebank. Above results manifest that our test have successfully construsted an recombinant of ompA gene from chlamydia trachomatis wild strain J serotype.