Modification And Optimization Of Human Interferon ?-2b Gene And Its Expression Vertification In E.coli

Increasing expression efficiency of exogenous genes is a hot topics in biotechnology.In theory,it is meaningful to modify and optimize an exogenous gene to obtain high expressed efficiencies in host cells.In our study,a new theoretical method was proposed to modify and optimize human interferon ?-2b gene(IFN ?-2b)and the gene was expected to obtain high expressed efficiencies in E.coli.There are two parts in our study.In first part,the protein coding sequence of IFN ?-2b gene was modified into the preference modes of E.coli high expressed genes,then the promoter region and the terminator region were selected so as to form a full gene.In second part,the optimized IFN ?-2b gene was expressed in E.coli to verified the reliability of our theoretical methods.Part one,the modification and optimization method of IFN ?-2b gene.There were two steps in modifying CDS sequence.First,all of the codons in CDS were substituted the optimal synonymous codons of high expressed genes of E.coli.Second,all of the base correlations in the third base of adjacent codons in the CDS were substituted into the strong correlation modes of high expressed genes of E.coli.In the process,we choose the optimal selection between the strong correlation modes and the optimal/second optimal synonymous codons.Then after,the promoter region and the terminator region of the highly expressed lpp gene in E.coli were selected as the promoter region and the terminator region of the IFN ?-2b gene.The SD segment in the promoter region of lpp gene was called high expressed SD,the SD segment inlow expressed genes of E.coli was selected and called low expressed SD.The high expressed SD was substituted by the low expressed SD to form the second promoter sequence as the contrast sequence.At last,three full genes were constructed.They are ‘high expressed SD+original CDS'(called GWGB gene),‘high expressed SD+modified CDS'(called GGB gene)and ‘low expressed SD+modified CDS'(called DGB gene).Part two,the GWGB gene and the GGB gene was selected as the first control group,the GGB gene and the DGB gene was selected as the second control group.Then,the transgenic experiments in E.coli were done by the two control groups.For the first control group,the aim is to verify the expression difference between the gene with modified CDS and the gene with original CDS in transgenic experiments.For the second control group,the aim is to verify the expression difference between the gene with high expressed SD and the gene with low expressed SD in transgenic experiments.The two methods were used in our transgenic experiments.(1)because the designed sequences is full gene sequence,the recombinant clone vector p GM-T-IFN?-2b was converted to E.coli to accomplish the expression process.(2)the recombinant expression vector p ET-28a(+)-IFN?-2b was converted to E.coli to accomplish the expression process(the promoter T7 in the expression vector was suppressed).(3)the ELISA method was used to detect the gene expression quantities of IFN?-2b genes in the two expression vectors.In the clone vector,the results showed that the expression quantities in the modified CDS gene(GGB gene)is significantly higher than in the original CDS gene(GWGB gene),the expression quantities increase 55.1%.The expression quantities in the high expressed SD gene(GGB gene)is significantly higher than in the low expressed SD gene(DGB gene),the expression quantities increase 52.8%.In the expression vector,the results showed that the expression quantities in the modified CDS gene(GGB gene)is significantly higher than in the original CDS gene(GWGB gene),the expression quantities increase 9.1%.The expression quantities in the high expressed SD gene(GGB gene)is significantly higher than in the low expressed SD gene(DGB gene),the expression quantities increase 18.2%.Results indicated that our theoretical method of gene modification and optimization were verified by the experiments.We think that our methods is meaningful to enhance the expression efficiency of exogenous genes in E.coli as well as in the other prokaryotes.