Expression Of Hepatocellular Carcinoma-Associated Antigen SMP30 MRNA On Human Tumor Cell Lines And Preparation Of Monoclonal Antibodies Against SMP30

Objective:To investigate the expression of SMP30 mRNA in tumor cell lines. To obtain a hybridoma cell line that stably secret monoclonal antibodies against senescence marker protain-30.Methods:RT-PCR and Real-time fluorescence quantitative PCR were used for detecting the expression of SMP30 mRNA in the normal liver cell, hepatocellular cancinoma cell, gastric cancer cell, breast cancer cell and cervical cancer cell. SPSS (13.0) statistical software was applied for data analysis.B lymphocyte hybridoma technique was applied to obtain a hybridoma cell line that stably secret monoclonal antibodies against senescence marker protain-30.Results:The expression of SMP30 mRNA in tumor cell lines of hepatocellular cancinoma, gastric cancer, breast cancer and cervical cancer by RT-PCR was 0.612±0.324,0.579±0.304,0.486±0.223,and 0.469±0.215, respectively, higher than that of normal liver cell (0.102±0.065). The differences have statistically significant (P<0.05). The expression of SMP30 mRNA in tumor cell lines of hepatocellular cancinoma, gastric cancer, breast cancer and cervical cancer by Real-time fluorescence quantitative PCR was 0.926±0.340, 0.922±0.379,0.614±0.356 and 0.608±0.346, respectively, higher than that of normal liver cell (0.175±0.158).The differences have statistically significant (P<0.05). SMP-30 fusion protein was expressed by the technique of genetic engineering and purified by affinity chromatographer. The titer of sera antibodies from 5 BALB/c female mice were greater than 1:16000 after they were immunized using SMP-30 protein. Cell fusion was made with spleen cells of mice and sp2/0 mouse myeloma cells. Five positive clones in fusion cells were obtained after screening by HAT and HT.Conclusion:The expression of SMP30 mRNA in cancer cells was higher than that of normal liver cell.The expression of SMP30 mRNA maybe has clinical application value.The developed method of the SMP30 monoclonal antibody was explored.Five positive clones in fusion cells were obtained. A platform for the follow-up study was provided.