| Cancer antigen（CA125）, which had been discovered and named byBast, et al in1983, is abnormally elevated in serum of malignant tumorpatients, especially of ovarian cancer patients. After its discovery, CA125has been extensively applied as a tumor marker for screening, auxiliarydiagnosis and therapeutic effect evaluation of ovarian cancer. At present,anti-CA125antibodies for clinical detection and basic research mostlydepend on expensive import, which seriously restricts application ofCA125in our country. Thus, it is valuable to develop a rapid, efficient andlow-cost method of anti-CA125antibody preparation for diagnostic kitdevelopment and relative functional research of CA125.Objective:1. To construct a rapid, efficient and low-cost prokaryotic expressionsystem of the tandem repeat of CA125(CA125R), to express, purify andidentify the recombinant CA125R protein.2. To prepare anti-CA125R antiserum after a rabbit was immunizedwith the recombinant CA125R protein, to purify the polyclonal antibodyand then, identify the specificity to natural CA125glycoprotein. 3. To prepare anti-CA125R monoclonal antibody by hybridomatechnique after BALB/c mice were immunized with the fusion protein, andidentify the specificity to natural CA125glycoprotein.Methods:1. A full gene of CA125R was synthesized and identified. This genewas cloned into pGEX-6P-1and pET-32a(+) prokaryotic expressionvectors to construct recombinant prokaryotic expression vectors:pGEX/CA125R and pET/CA125R, respectively.2. The recombinant plasmids pGEX/CA125R and pET/CA125R weretransformed into E.coli BL21(DE3) respectively. After the expressionconditions were optimized, recombinant protein expression was inducedand the fusion protein was purified through GST and Ni2+affinitychromatography separately. The pure fusion protein was identified byWestern blotting.3. A rabbit was immunized with the pure recombinant proteinTRAX-CA125R to prepare antiserum. The polyclonal antibody waspurified through Protein A antibody purification system and identified.4. BALB/c spleen cells and SP2/0cells were fused after BALB/c micewere immunized with prepared fusion protein GST-CA125R. The positiveclones were screened and further cloned to establish hybridomas whichcould steadily secrete anti-CA125monoclonal antibodies. The anti-CA125mAb was prepared by mouse ascites induce method and purified through protein A affinity chromatography, and identified.Results:1. The recombinant protein CA125R prokaryotic expression systemswere successfully established by gene recombination technology. Therecombinant protein GSTCA125and TRAX-CA125R were purifiedthrough GST and Ni2+affinity chromatography.2. After a rabbit was immunized with the pure TRAX-CA125Rprotein, anti-CA125R antiserum was prepared and purified. Westernblotting confirmed that the prepared polyclonal antibody could speciallyrecognize the natural CA125glycoprotein.3. The BALB/c mice spleen cells and SP2/0cells were fused by hybridtechnology after BALB/c mice were immunized with the pureGST-CA125R protein. One hybridoma was obtained which could steadilysecrete anti-CA125monoclonal antibody. The anti-CA125monoclonalantibody of high purity was prepared by ascites induce and furtherpurification.Conclusion:A rapid, efficient and low-cost prokaryotic expression system ofrecombinant CA125R protein has been successfully established by generecombinant technology. The recombinant GST-CA125R andTRAX-CA125R protein with high purity are prepared. Rabbit anti-CA125polyclonal antibody is prepared which can specially recognize natural CA125glycoprotein. One hybridoma that can steadily secrete anti-CA125monoclonal antibody has been successfully obtained and anti-CA125monoclonal antibody of high purity is prepared. This lays some foundationfor relative functional research and development of clinical detection kit ofCA125. |
PDF Full Text Request