Modification Of Human Interleukin-2 Gene Sequence And Verification Of Its Expression In E.coli

This paper mainly studied two aspects.First,the sequence of human interleukin-2 gene was modified and optimized.Firstly,the coding sequence(CDS)of human interleukin-2 gene was transformed into the high expressed pattern of E.coli,and the ribosomal binding site(SD)sequence of the highly expressed gene in E.coli was selected.The SD sequence of the low expressed gene in E.coli was also selected as a comparison.Then it was assembled into "high expressed SD + modified CDS" and "high expressed SD + original CDS" gene control group,"high expressed SD + modified CDS" and "low expressed SD + modified CDS" gene control group.The former mainly investigated the effect of modified CDS on gene expression,while the latter mainly examined the effect of optimal selection of SD sequence on gene expression.Second,the assembled two groups of gene sequences were cloned and expressed in E.coli TOP10,and the theoretical prediction of the modified and optimized expression level of human interleukin-2 gene(hIL-2)was verified by experiments.It was speculated that the expression level of the modified hIL-2 gene coding sequence in E.coli was that the expressed level of CAI=0.884,original hIL-2 gene coding sequence in E.coli was CAI=0.267.In order to ensure the normal initiation ofcoding gene expressed,the promoter region and Terminator region of the highly expressed rplL gene in E.coli were selected to replace the promoter region and Terminator region of hIL-2 gene.The high expressed SD sequence was the SD sequence of the gene,and the low expressed SD sequence was the SD sequence of the low expressed gene given in the previous study to replace the SD sequence of the rplL gene.The gene expression was carried out by recombinant expressed vector pBV220/hIL-2.In the experiment,SDS-PAGE was used to characterize the expressed protein,and then ELISA was used to quantitatively detect the expressed protein induced by 42 ? for 3 h,4 h and 5 h,respectively.The results showed that: Part one,in the control group of "high expressed SD + modified CDS" and "high expressed SD+ original CDS" gene control group,the protein expression after CDS modified was significantly higher than that before CDS modified;The protein expression was increased by 131.41%,and the experimental results were in good agreement with the theoretical prediction,which indicated that the protein expression of the modified coding region was significantly higher than that of the original gene.Part two,In the gene control group of "high expressed SD + modified CDS" and "low expressed SD+ modified CDS" gene,the protein expression of the gene corresponding to the high expressed SD sequence was significantly higher than that of the gene corresponding to the low expressed SD sequence.The expression of protein increased by 36.55%,indicating that the high expression of SD sequence itself can improve the level of gene expression.The experimental results verify our theoretical prediction.In a word,by modifying the gene coding sequence and optimizing the SD sequence in the gene promoter region,the protein expression level of foreign genes in host cells can be significantly increased.The research methods in this paper have important reference value for improving transgenic efficiency.