Study On The Expression And Purification Of Chlamydiaphage Capsid Protein Vp1

Chlamydia trachomatis (Ct) is one of the most common venereal diseasepathogen internal and abroad. It is also the main pathogen of NGU. Urogenital Ctinfection can cause many diseases .Except for NGU, it causes many complications.Chlamydiaphages are the type of microvirus,the major component of the capsid isVp1,which is conservative and special.It is important in finding otherchlamydiaphages and possible in the process of implantation .The recentresearchments have just showed us the gene ,constructure and molecular characters ofchlamydiaphage.The work we have done is the purification and expression ,andsettling the foundation for the next step.Objective: To get the Chlamydial GPIC Capsid Vp1 protein which is highlyconserved and important for studying Chlamydial infection.Methods: Select one colony which grows on the plate with antibiotics, cultivateit in LB medium,and extract its plasmid.Sequence the plasmid to confirm the vp1gene have been cloned correctly.Induce the E.coli with the plasmid to get the targetprotein.By SDS-PAGE,we can recognize the target protein is in the surpernant orsediment.During western blot we use the histag antibody as the first antibody and thenconfirm the protein is Vp1. Optimizating the conditions of inducing,get more targetprotein.Resolve the protein with the binding buffer(contain 6Mureal),then put theliquid in the column contained the his-binding resin.Washing the column step bystep,collect the liquid which may contain the target protein.By western blot,confirmthe protein is the Vp1 protein.Choose the liquid which show only one band afterSDS-PAGE,put them together, then determine the content of the protein.Renaturate the protein by dialysis,then identify the immunocompetence of the protein with Vp1antibody.Results:.The plamid contain the correct sequence of the vp1 gene.After beingreduced,SDS-PAGE and Western Blot both show that we obtain the Vp1 protein in thesediment which is about 70KD.During the purification we get the protein sampleswhich contain a single band by SDS-PAGE.Use the Vp1 antibody as the firstantibody, by western blot,we get the band and identify the renaturate d protein hasimmunocompetence.Conclusions: 1.The recombinant plasmid conclude the correct gene of Vp1protein. 2.After being induced,the recon expresses the Vp1 protein. 3.During thepurification we get the protein that show the single strap by SDS-PAGE4.After therenaturation the Vp1 protein we obtain shows the immunocompetence.