Expression And Purification Of The Recombinant Abrin-A A, B Chain Protein In Escherichia Coli And Its Biological Activities

In this experiment, to improve levels of expression, the gene encoding ABRaA was firstly optimized by replacing rare codons with high-frequency ones, and then was synthesized using two-step PCR (0.75kb) . Subsequently the synthesized gene was respectively constructed into the expression vector pMBP-p pET-Dsba pET-Trx and pET-His, and expressed in Escherichia coli cells. Among resulting recombinant expression vectors, an optimal cytoplasm expression in soluble form was acquired by the expression vector pET-HisA, and it occupied 30% of total cell protein. The recombinant protein (rABRaA) was one-step purified by Ni-NTA metal-affinity chromatography, and its purity is up to 98%. The Western blot showed that rabbit polyclonal antibody against native abrin antibody has specific affinity for rABRaB. The purified rABRaA depurinated rat liver28S rRNA atA4324, and after treated by aniline, a 420nt fragment was generated from 28S rRNA. The result showed that the rABRaA possesses N-glycosylase activity. The purified rABRaA was tested for its biological activity by measuring the ability to inhibit the protein biosythesis of rabbit recticulocyte cell-free system, The concentration at which L-[3H]-leucine incorporation was inhibited by 50% was 8.5x10-11M. In particular, the MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. The IC50 is 5.2x10-6and 5.8x10-7M.In addition, total RNA was isolated from the plumule of Abrus precatorius, by reverse transcription polymerase chain reaction techniques, the gene of ABRaB was derived and cloned into the pGEM-T and its nucleotide sequence was determined.Subsequently, the ABRaB was subcloned into the expression vector pET-28a, and expressed in cytoplasm of Escherichia coll BL21 cells. The relative molecular weight of target protein was about 35kDa, similar to the ABRaB, and the target protein occupied 11% of total cell protein, mainly in inclusion bodies. The Western blot showed that rabbit polyclonal antibody against native ABRaB antibody has a specific affinity for rABRaB. The inclusion bodies were washed and then one-step purified by Ni-NTA metal-affinity chromatography, and its purity is up to 98%. The rABRaB was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against rABRaB were generated by using sarcoma 180 cells, and the antibody titer of ascites was up to 2 10. The results laid the foundation for the detection of abrin and genetic vaccine development.