Study On The Existence Of GPIC Phage Capsid Protein Vp1 Antibody In Sera Of Chlaydia Trachomatis Patients

Chlamydia trachomatis is the main pathogens causing Genital chlamydial infection accounted for 40%～50%.Patients have symptoms or not,so diagnosed difficultly.Chlamydia trachomatis infection can cause prostatitis,epididymitis, proctitis and other complications,resulting in serious consequences.At the same time drug resistance increased the difficulties of treatment.Therefore,Chlamydia phage research has been concerned about.Bacteriophage is a virus,which can make the bacteria disintegrated and can be widely used to block the infection,so anti-infective phage research has been extensive attention.This topic is designed to GPIC phage capsid protein Vp1 expression,identification,purification.Do screening of the serum antibody Vp1 among the collection of 206 cases.It's very important to make sure the existence of bacteriophage for the treatment of Chlamydia trachomatis.Objective:Attain the GPIC phageφCPG1 capsid protein Vp1.Collect some cases of Chlamydia trachomatis infection of the serum in order to discover the antibody of Chlamydia phage Vp1 protein,which can promote Chlamydia trachomatis further research to and phage.Methods:Choose a prokaryotic expression plasmid Vp1-pET-30a(+) recombinant plasmid bacteria to inoculated in LB liquid medium for induction of expression,partially purified,centrifugal collection of insoluble protein.By protein purification kit instructions,the purified Vp1 protein can be analysis by polyacrylamide gel electrophoresis.Apply of coomassie brilliant blue method to quantitative analysis projected Vp1 protein concentration.Use the renaturation Vp1 protein immunized in mice with 3 times.Then collect blood serum from the inner canthal vein.Vp1 is coated enzyme-linked protein in 96 crates in 4℃overnight, After PBST washing,3%BSA containing,do ELISA among 84 cases of normal and 206 infected C.t serum samples to test the OD₄₀₅.Take Western blot with purified Vp1 protein,and transfer it to nitro-cellulose membrane.Then after 3%milk-TBST solution closed,the immune serum,not infected with C.t serum,ELISA-positive results serum are added with 1%milk-TBST 1:500 dilution for overnight incubation.TBST washing,add alkaline phosphatase labeled goat anti human IgG antibody(1:10000 incubation).After BCIP/NBT color reaction,use the termination of water and observe the color change toVp1 antibody identification.Results:The Vp1-pET-30a(+) is induced and expressed.After Polyacrylamide gel electrophoresis,there is a clear band about 62,000,and it is Vp1 protein.Do dialysis and renaturation about the purification purpose protein,so that Vp1 protein is antigenicity,use Coomassie brilliant blue method to get concentration.Purified Vp1 protein was coated plate with 100ug/well.Immune serum,normal human serum were incubated with different dilution conduct.Then we find that of the positive serum OD₄₀₅/negative serum OD₄₀₅ in 1:400 dilution=2.5496,so this ratio is the biggest difference.Ultimately the optimum serum dilution is 1:400.The tested serum samples whose OD₄₀₅ is more than 0.102 are positive,and the contrary are negative.Among 206 cases,there are 18 people showed the positive result,and their serum antibodies may have Vp1.Doing Western blot on the 18 ELISA positive serum,we discover four cases of serum antibody with Vp1 suggesting that the Vp1 antibody exist in serum of Chlamydia trachomatis infection patient.Conclusion:1.Successful expression,identification,purification of the protein Vp1 of Chlamydia phageφCPG1 2.Collection of Chlamydia trachomatis infection serum,and through enzyme-linked immunosorbent assay(ELISA),discovering 18 cases having antibody to protein Vp1 in serum of selected cases of Chlamydia trachomatis infection.3.Identification the existence of Vp1 antibody through Western blot,and 4 cases show the positive results.