| Chlamydia has increasingly become an important pathogen of human diseases, which has caused a worldwide scope of the trachoma widespread, causing serious harm, and so far it is still the leading cause of blindness in a number of developing countries . Phage is a group virus specifically to infect and schizolysis the host bacterias. 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. Chlamydia phage is a microvirus, currently only found to exist in several Chlamydias such as Chlamydia pneumoniae, Chlamydia abortus, and Chlamydia fishers, and studies have shown that the shell of chlamydia phage is composed of capsid protein, whose main ingredient is protein Vp1, Vp2 and Vp3, Vp1 is the main structural protein. These three proteins may have an important role in the Chlamydia phage adhesion and plant to the Chlamydia . Meanwhile, the protein is highly conserved and specific, so it is a good marker for other Chlamydia species - particularly Chlamydia trachomatis to find Chlamydia phage. This subject seeks to express and purify Vp1 protein, to study the immunogenicity of this protein and to prepare the monoclonal antibody. This lay the foundation for further stuties of the clinical significance of monoclonal antibodies, and the relevant mechanisms of the interactions between Vp1 protein and Chlamydia. Through the purified Vp1 protein and the resultant monoclonal antibody , we can do the screening trying to find the phage of Chlamydia trachomatis .OBJECTIVE: to express and purify the Vp1 protein of the Chlamydia phage, and to prepare the monoclonal antibody.METHOD: Escherichia coli BL21 carrying vp1 - pET30a(+) , after the plasmid sequencing, and screenig the Kanamycin antibiotic, was inducted through IPTG for expression. With the help of trixton100, the Escherichia coli was schizolysed in the 6M urea buffer solution and sonicated. After the cell wall was schizolysised, the protein was dissolved in the 6M urea buffer solution and filtrated by 0.45um aperture filter. The protein, after being centrifuged and resuspened , the liquid became clear. Then after dialysised by the nickel ion dialysis column, the Vp1 protein is bond on the dialysis column with the nickel ion and was not filtered. With binding buffer and washing buffer, cleaned the dialysis column in turns. The mixed protein was eluted and finally using strite buffer, the Vp1 protein binding on the nickel ion could be eluted after renaturation. Then we can obtain the sole strap in the the band size of 70KD in the SDS-PAGE electrophoresis.The protein quota about the Vp1 after renaturation. For the immunization of the BALB / C mice, we use approximate 70μg of protein to inject the 4-week-old BALB / C mice intraperitoneally . One month later, we took the angular vein blood for the Western-blot, with the goat-anti-mouse monoclonal antibody as the second antibody. In the size of the molecular of Vp1 protein there showed a clear band. Took the mice spleen cells after supplement immunity three days before, with myeloma cells after 10:1 quantity mix. After 4000 r, under the conditions in 37℃water , add the 50% PEG 0.4ml to the cell precipitation by drops with the sucker, and mix evenly, for 90s. Then after 4000 revolution of centrifugal 4 minutes, add the complete culture solution to the precipitation, stop the response of PEG .Then centrifuge the cell suspension by 4000 revolution for 4 minutes, add the fusion cell to the 96 pore plate concluding the mouse abdominal cavity macrophage. And put the plate in 37℃thermostat including the carbon dioxide . Take the supernatant of the hybridoma cell after HAT, HT screening to do the ELISA, to test whether there is the specific antibody.RESULT: Escherichia coli BL21 carrying vp1 - pET30a(+), inducted through IPTG for expression, had shown that we could get the Chlamydia GPIC Phage protein Vp1 about 70 KD size from the precipitation of the lysate by SDS-PAGE and Western-Blot. The protein after purification in the SDS-PAGE showed a single band, indicating the purified Vp1 protein containing less miscellaneous protein. After immunizing the mice with the purified protein, We took the angular vein blood for the Western-blot, with the goat-anti-mouse monoclonal antibody as the second antibody. In the size of the molecular of Vp1 protein there showed a clear band with the DAB as the developer, indicating that the surum of the mice after immunitization can produce the antibodies against the Vp1 protein. Making the mice spleen cells after supplement immunity fusion with the myeloma cells, can form hybridoma cells, which can secrete some specific antibodies.CONCLUSION: 1,Escherichia coli concluding the plasmid after the induction can express the capsid protein Vp1 of Chlamydia bacteriophage, which can be separated and purified successfully. 2,Vp1 recombinant protein has the immunogenicity. 3,Using the capsid protein Vp1 of Chlamydia GPIC bacteriophage after renaturation to immunity BALB/C mouse , the surum of the mice can produce the antibodies against the Vp1 protein . 4,The spleen cells of the immunitied mice can make fusion with the myeloma cell under the help of the feeder cell .The fusion hybridoma cells can be cultured in vitro and can secrete some specific antibodies. |
