Construction Of Prokaryotic Expression Of Omp1 For Chlamydia Trachoatis E Serotype And It's Expression And Purification In Prokaryotic Cell

Infection of human urogenital tract caused by Chlamydia trachomatis(C.t) is more and more these years.Asymptomatic and chronic C.t infection may result in severe complications such as prostatitis,infertility,ectopic pregnancy and chronic pelvic inflammatory disease etc.C.t is remarked for its state of protraction, refractoriness,easy relapse,and striking consumption.Study on C.t infection has been one of the popular topics of the world.It's urgent to improve the prevention and control measures for C.t prevalence.Research on C.t vaccine is the key point of prevention measures.Research has indicated that Chlamydia trachomatis major outer membrane protein epitope decided to produce protective antibodies,and specially as the target antigen of the antibodies.MOMP,its molecular mass about 40×10~3,is the major components of Chlamydia outer membrane complex and about more than 60 percent in the total outer membrane protein.Objective:To clone omp1 gene from genomic DNA of Chlamydia trachomatis serotype E and to construct prokaryotic expression plasmid of omp1-pGEX6p-1,and achieve the fussion expression in the coli(BL-21).Moreover,to obtain the purified and quantitated MOMP.Methods:Extracted DNA sequence of C.t E serotype which coding MOMP as a template,designing primer and amplifing the ompl genes according it.Omp1 and pGEX6p-1 were linked to be recombinant plasmid omp1-pGEX6p-1,transforming into E.coli BL21.Select one colony which grows on the plate with antibiotics, cultivate it in LB medium,and extract its plasmid.Recombinant plasmid is identified by Enzymes cutting,PCR amplification and sequencing respectively,and the result is right.Induce the E.coli with the plasmid to get the target protein.By SDS-PAGE, we can recognize the target protein is in the surpernant or sediment.During western blot we use the GST-tag antibody as the first antibody and then confirm the protein can be detected.Also the recombinant plasmid is identified by PCR.After that,the protein is purified by GST resin and identified with 1%agarose gel electrophoresis.Then,the MOMP was quantitated by Elisa,and to evaluate the MOMP quantity with data analysis. Results:The sequence of gene which got from PCR are similar with omp1 of C.t E serotype in Genebank about 99.92%,showing that we have got omp1 of C.t E serotype.After enzymes cutting,we got two parts of gene seqeuence,1200bp and 5000bp separately.We can draw a conclusion that recombinant protein MOMP of C.t E serotype was constructed successfully after digestion,PCR and sequence identity The target protein induced is about 66KD and can be detected by GST-tag antibody. PCR of recombinant plasmid showed that the protein is right.There is only one strip adout 66KD with 1%agarose gel electrophoresis after GST resin.The high concentration MOMP is gained after purification.Conclusions:(1)Omp1 gene of C.t E serotype has been cloned and recombinant omp1-pGEX6p-1 has been constructed successfully;(2)The protein recombinant plasmid expressed can be detected by GST-tag antibody;(3)Sequence of recombinant plasmid and the following mice trial showed that the taget protein has immunogenicity theoretically.