| It has been indicated by many research that the occurrence and development of liver cancer is closely linked with the mitochondria, and aflatoxinB1 (AFB1) is one of the most important external factors that induce liver cancer. AFB1 not only make the mice with progressive weight loss and caused liver cancer, but also can induce or inhibit the expression of some proteins of the liver through two-dimensional electrophoresis and mass spectrometry analysis methods in previous study, idh3αis one of the critical protein. Antibodies made in this study will be used in Western-blotting for verifying the IDH expression levels in mammalian cells under different environments, and for finding the proteins that are interactive with IDH protein by affinity chromatography and immunoprecipitation methods.This study first built the idh3αonto the pET-32a to get the recombinant which was then transformed into E.coli BL21 (DE3) in the host bacteria. Induced by IPTG, The positive bacteria successfully expressed the fusion protein. The purified protein, which was used for immunization of BALB/C mice, was picked up Ni2+ column. After a certain potency of mouse spleen cells were isolated and monoclonal antibody preparation and extraction of spleen RNA genetic engineering scFv.Fused the spleen cells of mice immunized with the myeloma cell SP2/0 to form hybridomas, HAT culture medium screening, ELISA test, positive hybridoma cloned three times, testing, screening out a stable secretion of high affinity and specificity of cell line, after purification and properties of identification ,we received McAb.RNA was reverse transcripted to cDNA and amplificated into scFv's VH and VL , which was then assemblied into scFv genes by a flexible peptide, then this genes were subcloned into phage vector on the establishment of antibody phage libraries, enrichment panning, selected a stable high-affinity and specificity of recombinant, then after soluble expression, purification, identification, scFv can be prepared. |
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