The purification and primary application of rabbit anti-hEra polyclonal antibody

Abstract: era gene is an open reading frame which located downstream of the rnc gene encoding ribonuclease III.It was originally named on the basis of partly similarity to Ras of human and yeast. Genetic experiments have shown that era is a key gene for cell cycle and cell division. C. Slogans -, Antirrhinum^ mouse and human have genes that had homology to era gene. The full length of human era-cDNA gene is 2.2 kb and contains an open reading frame of 1038 bp in length that encodes a protein of 346 amono acids. It contains N-terminal GTPase domain and C-terminal KH domain .However, It is not clear on function of human Era by now.In order to investigate the function of era gene which is highly conservative from E. coli to human, we engage in the following basic experiments:Rabbit anti-hEra polyclonal antibody was prepared by immunizing rabbit with hEra C-terminal domain protein which was cut from SDS-PAG and grinded into gel suspension liquid. The titer of polyclonal antibody can reach 1:800 through Western blot analysis. The engineering strain which contains a recombinant expressed plasmid pMSM-hEra was induced with 0. 3mmol/L IPTG . The expression level of MBP-hEra was increased with the prolongation of time. It can reach the maximum by 4hr and constitute 23.9% of total cell proteins. The engineering strain was lysised by ultrasonic wave. Much of MBP-hEra existed in the supernatant of lysate in a soluable form. MBP-hEra was purified through amylose resin chromatography column and Superose 12 gel filtation. The purity of MBP-hEra could reach about 95%.The total rate of recovery of MBP-hEra is 69%. The purified MBP-hEra was coupled to the NHS-activated sepharose?to prepare affinity chromatography column to purify rabbit anti-hEra polyclonalantibody. The purified polyclonal antibody had better specificity than the nonpurified polyclonal antibody throuth Western blot analysis. In addition, The result of Western blot analysis also showed that the hEra was expressed in different fetal tissues such as kidney > liver> lung> muscle and thymus. At last, hEra was detected in the hepatocellular carcinoma of 54 cases by immunohistochemistry. The results showed that the expression level of hEra in hepatocellular carcinoma was much more than that in the normal liver tissue. The clinical data also showed that the expression level of hEra was highly correlated with differentiation. So we can draw a conclusion that the detection of hEra could be used as a predictor for dignosis and prognosis of hepatocellular carcinoma…
Key words: rabbot anti-hEra polyclonal antibody; purification; hepatocellular carcinoma

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