A Study On The Gene Engineer Expression Of Human Interleukin 3 Gene And Isolated And Purification Recombinant Protein

Human interleukin 3(hlL-3) is a key regulatory molecule in the early differentiation of hematopoietic precursor cell. We have amplified a 0.44Kb DNA from cDNA Libraries of T cell by PCR and cloned it into the plasmid vector pUC19. DNA sequencing showed this DNA fragment encompassed the complete hlL-3 coding region and had Nco I site and ATG code at 5' end. The expression plasmid of hlL-3 gene under the control of λ P_L promoter was constructed and transformed to E.coli Tap106. The hlL-3 protein product was obtained from the cell induced at 42℃ and was about 15% of total cellular proteins. In order to improve the expression,the 5' end sequence of hlL-3 cDNA and translation initiation region (TIR) was mutated by a serious of synthetic primers.And the expression level of hlL-3 was increased to 30%of total cellular proteins with the yield of 24.4g/L of bacterium. A simple and effective method of isolation and purification was developed. The purity of hlL-3 was more than 95%. The specific activity was more than 1.2×10⁷ u/mg and total recover was 28.2%. The recombinant hlL-3 was further identified by ELISA,western-blot, isoelectric focus and amino acid sequencing of N-terminal 16 a.a.. In this paper,we also studied the effection of bacteriophage T₇ gene 10 translational enhance sequence on the expression of hlL-3 in E.coli, the secretory expression of hlL-3 in yeast and the fusion expression of hlL-3 and hlL-11 gene in E.coli.