Study On The Expression And Role Of Protein-protein Of Chlamydiaphage Capsid Protein Vp1

Chlamydia trachomatis is the most popular cause of sex trasnmited diseases in the world. Phages is a kind of virus that can infect bacterium, fungus, actinomycetes and spirochaetes etc,and becoming more important in Molecular Biology and as a Antimicrobial.Phage can be divided into two groups, and some studies have shown that the shell of chlamydia phage is composed of capsid proteins, whose main ingredient are protein Vp1, Vp2 and Vp3. Vp1 is the main structural protein. We have the phiCPG1 Vp1protein,and if we have the other type of the protein we can study the phage of host macrophages, interaction between the Chlamydia and the phage,and the design of molecular tools.In this researchment we express and purify the Chlamydiaphage Chp3Vp1 protein; observe the in vitro interaction of Chlamydia protein; preliminary explore the multiple epitopes design and function in capsid protein.To get the codon optimized nucleic acid sequences according to Chp3vpl by bioinformatics analysis ues the overlapping extension and/or the recombinant gene synthesis technology,and the E. coli BL21 with the recombinant gene after antibiotic kanamycin selection from single colony culture is raised and the extracted recombinant plasmid is sequenced and compared with the vpl sequence of the Genebank(GeneID:1261929).To express the protein with IPTG.After the cntrifuge the bacterial lysate supernatant and precipitation were performed Westernblot, while anti-his tag antibody (Histag antibody) as the primary antibody,With the Optimization of IPTG concentration induction time express the protein,purify the protein by SDS-PAGE gel,and quantify the protein by Coomassie brilliant blue method. To detect the protein-protein interaction of recombinant protein and Momp/Pomp protein.To design the protein with multiple epitopes by bioinformation method,identify and purify the protein.Single digestion of recombinant plasmid shows a single about 8000bp DNA bands, sequencing results show than the homology of 99.46% for the synonymous mutation and amino acid homology is 100% correct. Induced by IPTG, SDS-PAGE electrophoresis and Western Blot show about 55kD a band from the centrifugation but there is no the same band from the supernatant.The Optimized induction conditions are 370C,0.3mM IPTG and induced for 2 hours.The concentration of the protein is 126.3ug/ml.Using polyclonal antibodies as the first antibody,the Westernblot result show the 55KD band.The result of Far Westernblot show the protein about 40KD from the Chlamydia supernatant can be identified by the Chp3Vp1 protein,but Pomp and Momp cannot be.With the analysis of the Chlamydiaphages Vp1 protein epitopes,we design the protein,and analysize the protein then get the similar results.After inducing the protein,we identified the band about 40KD,and the optimized conditions is 370C,0.08mM IPTG 4 h. The induced bacterial protein diluted 640-fold, can be seen as a single target identification strip in Westernblot.We can get the following results:1. recombinant plasmid is correct,the induced protein can be identified as the form of inclusion bodies,and the Vp1 protein was initially separated successfully;2.there is the interaction between the Chp3Vp1 protein and the Chlamydia protein in the supernatants,which is about 40KD.But there is no interaction to Pomp/Momp protein;3.the designed protein has the similar epitope distribution and composition as the natural protein;4.the induction of designed protein is successful,and have got the preliminary pufied protein.