|
Chlamydia (Chlamydia) has increasingly become an important pathogen of human disease, which has caused the prevalence of trachoma worldwide, causing serious harm, is still by far the leading cause of blindness in some developing countries. Phage (bacteriophage; phage) is a group capable of specifically infecting host bacterium, and its cleavage of the virus can be, it is only in the study of molecular biology may play an important role in the treatment as an antibacterial agent, the bacterial infection is increasing attention. Chlamydia phage is a micro virus found in Chlamydia pneumoniae, abortion, chlamydia, fish Chlamydia several species such as Chlamydia, at present only the presence of bacteriophages, studies have shown that the main component of the composition of Chlamydia phage capsid protein shell to Vp1, Vp2 and Vp3, wherein Vp1 is the major structural protein, may have an important role in the phage chlamydia adhesion and implants. Meanwhile, the protein is highly conserved and specific, and thus look for other Chlamydia species - especially Chlamydia trachomatis the Chlamydia phage good markers. The purpose of this project is to study the separation and purification of Vp1 protein immunogenic to explore preparation Vp1 protein monoclonal antibody to lay the foundation for further study of its monoclonal antibodies of clinical significance and the Vp1 protein and Chlamydia mechanisms, and through purified the Vp1 protein and resulting Vp1 mAb for clinical screening, trying to discover the presence of Chlamydia trachomatis phage. Objective: Expression, purification Chlamydia phage the Vp1 protein, mice immunized with preparation of hybridoma cells. METHODS: Cultured E. coli BL21 recombinant plasmid with vp1-pET-30a () expression was induced by IPTG, ultrasonic broken bacteria, dissolved in 6M urea. Centrifugation, the resuspended protein was filtered through a 0.45μm pore size filter, the solution is clear, and then dialyzed through a dialysis column containing nickel ion, In use bindingbuffer washingbuffer sequentially cleaning dialysis column, eluting contaminating proteins, and finally will be combined in the nickel ions with stritebuffer on Vp1 protein eluted, renatured identified by SDS-PAGE electrophoresis. Vp1 protein refolded protein, approximately 70μg protein injection at the 4-week-old BALB / C mice were immunized intraperitoneally, after January whichever the inner canthus venous, as an anti to the sheep anti-mouse IgG secondary antibody, and Western-blot. Taken after the booster immunization days mouse spleen cells were mixed with myeloma cells in a quantity of 10:1, for cell fusion, the fused cells were added to 96-well plates containing mouse peritoneal macrophages in Incubate 37 ° C for carbon dioxide-containing incubator. Taken after HAT, HT screening of hybridoma supernatants, ELISA, detecting whether or not containing a specific antibody. Results: with vp1-pET-30a () recombinant plasmid by SDS-PAGE and Western Blot, are displayed can be obtained from the bacterial lysates were pelleted by centrifugation the Chlamydia the GPIC phage capsid protein of approximately 70kD coli BL21 induced by IPTG Vp1, was purified on SDS-PAGE can be displayed a single band, the purified VP1 protein purity. Western-blot at 70KD size, the clear purpose of the strip, which shows anti-Vp1 protein antibody contained in the serum of mice after immunization. Taken to strengthen the spleen after immunization, fused with myeloma cells to form a hybridoma cell, and capable of secreting the antibody. Conclusion: 1, containing vp1-pET30a () Chlamydia phage Vp1 capsid protein capable of expressing the recombinant plasmid of E. coli after induction. 2, Vp1 protein separation and purification success. 3, after purification Vp1 protein having immunogenicity, immunized mice with Vp1 for Vp1 protein capable of generating antibodies in the mouse serum. 4, the spleen cells of the immunized mice with myeloma cells for cell fusion, the fused hybridoma cells can be performed both in vitro culture, and capable of secreting a specific antibody of Vp1.
|