Preparation And Preliminary Application Of Monoclonal Antibody Against Vp1 Protein Of Chlamydiaphage PhiCPG1

Chlamydia trachomatis can infect many parts of the body, including eyes and Genitourinary tract. So far trachoma is still the leading cause of blindness in a number of developing countries. The infection of Genitourinary tract, which is more widespread, can leading to severe complications such as ectopic gestation, infertility, infant pneumonia and Reiter's syndrome etc.Phage is a kind of virus that can infect bacterium, fungus, actinomycetes and spirochaetes etc. Phage can be divided into two groups, one is virulence phage which can split host bacteria and the other is lysogenesis phage whose gene can integrated into the chromosomal of its host bacteria. it not only plays an important role in the study of molecular biology,but also calls increasing attention as antibacterial agents treatment of bacterial infections.Phage can infect chlamydophila, although it is obligate parasitism. So far,6 chlamydia phages have been found-Chp1,Chp2,Chp3,Chp4,ΦCPAR39 and PhiCPGl. Studies have shown that the shell of chlamydia phage is composed of capsid proteins, whose main ingredient are protein Vpl, Vp2 and Vp3. Vpl is the main structural protein. These three proteins may play an important role in adhesion and implantation to the chlamydophila. Meanwhile, the protein is highly conserved and specific, so it is a good marker for other Chlamydia species to find Chlamydia phage.To screening for phage in Chlamydia trachomatis, Vp1 protein was extracted and purified. BALB/C mice was immunized with Vpl protein. After 4 times of immunization, the mice's spleen was removed and fused with myeloma cells. The secration wells were selected with ELISA and sub-clone was done. Hybridoma cells and monoclonal antibody were identified after 3 times of sub-clone. Monoclonal antibody was prepared in quantity after hybridoma cells was injected into the abdominal cavity of BALB/C mice and purified by Protein G Sepharose 4 Fast Flow.McCoy cells were grown on 13mm coverslips in 24-well trays. Cells were infected with clinical isolates of Chlamydia trachomatis(20 isolates). After 48h post infection, the culture medium was removed, monolayers washed twice in PBS and fixed in ice cold methanol for 15 min. Monoclonal antibody was incubated with fixed cells for 40min at 37℃, washed three times in PBS. Bound antibody was detected with anti-mouse fluorescein conjugated antibody diluted in 0.0025% Evans Blue dye in PBS.The serum antibody titre can reach 1:6400 after three times of immunization.3 secration wells were obtained after fusion. Monoclonal antibody was prepared after 3 times of sub-clone and identified as IgG1(κ). Chromosome analysis of hybridoma cells showed the mean number was 98, most of them were telocentric chromosome and a few were submetacentric chromosome. The titre of monoclonal antibody purified was more than 1:102400.20 clinical isolates were screened by the monoclonal antibody and no positive results.This study successfully got the purified protein vpl and prepared the monoclonal antibody to the Vpl protein of Chlamydia phage PhiCPG1.20 Clinical isolates were screened by immumofluorescence method but got no positive result.