Recombination And Expression Of Liver Cancer Associated Antigen CNN2Andserum Analysis

OBJECTIVE:To obtain and purify hepatocellular carcinoma-associated antigen H2-calponin (CNN2) fusion protein with a6×HIS tag. To study the expression of CNN2antibody in HCC patient’s serum.METHOD:PCR amplification CNN2cDNA sequence, the cDNA of CNN2was amplified and recombined in expressing plasmid(pCold Ⅲ). The optimal expression condition was explored (such as temperature, time, concentration and so on), and HIS-CNN2fusion protein was expressed in E.coli BL21(DE3)pLysS and purified by Ni-NTA resin. The method of WB was used to identify CNN2protein. The optimal ELISA experimental conditions was explored and established, and then175patients with liver cancer was detected their serum autoantibodies to CNN2by the methods of indirect ELISA.RESULT:CNN2recombinant was connected into DH5a Escherichia coli,The strain can be growth up in LB plate with Amp, Purpose gene can be amplified in bacterium fluid by PCR.CNN2recombinant was showed two bands with1000bp and a large of plasmid strip when recombinant plasmid was cut by double enzyme Nde Ⅰ and Sal Ⅰ. Finally the sequencing was blasted with sequence on NCBI, the similarity was100%; Using the optimization expressing conditions would got the largest amount of protein. the optimization expressing conditions was that, cultured in37℃for2.5h before added inducer,the concentration of IPTG was0.6mM, bacteria expressing in25℃for7h. SDS-PAGE showed CNN2protein appearing in souble and precipitate parts of bacteria. Expression level of interest protein(HIS-CNN2) was30%; WB show interest protein could combine with CNN2antibody; CNN2antibody was detected in HCC patients’ serum The positive expressed of CNN2antibody in HCC patient’serum was13.14%.CONCLUSION:The CNN2-pCold III recombined expression plasmid was successfully constructed. The optimization expressing conditions were0.6mM inducing concentration of IPTG, bacteria expressing in25℃for7h. The positive expressed of CNN2antibody in HCC patient’ serum was13.14%.