Studies On The Design,Synthesis And Expression Of The Mutation Of The CMIV

The Contents of this studies include:1) According to the researches on the correlation between the function and structure of the CMIV from Bombyx-moxi before by others,especially by Lixinlal in Naigin normal university of China,we have designed and Sythesized the mutation I of the gene of CMIV that was different from the natural CMIV about 50% in amino sequence,using the favorable Condon of the Ecoli. After cheked the result of Synthesis by sequence,We have cloned the gene into 3' of the gene of Thioredoxin in the Thio-Fusion Expression Vector(PTXFUS),and the fusion protein of Thio-CMIV was highly expressed in soluble form. Because of the cleavage site of Enterokinase and CNBr was designed in the middle of Thioredoxin and CMIV,the expressed Peptides of the Mutation of CMIV Could be Cuted down from the fusion Protein by Enterokinase or CNBr. 2)Basis of upon studies,we have also designed and Sythesized the Mutation II of the CMIV that been greatly changed in the 3' of the gene Comparing With nature CMIV:The hTGF3(The third loop region of hTGF2 Specifically binding to EGFR receptor) Was fused to 3' of gene of CMIV through a flexible linker. The gene of the Mutation II of CMIV was Sequenced and clonged to the vector of PTXFUS to fuse to the 3' of gene of thioredoxin. The SDS-PAGE results showed the fusion Protein was efficiently expressed in the soluble form. 3)The expressed fusion proteinwas purified and cleavaged by Enterokinase to release the Mutation I of CMIV and the Mutation II,whiches exhibited antibacterial activity to the Ecoli.K12 . 4)The results of this studies showed that we could produced the Mutation I of CMIV and the Mutation II of CMIV by fusion to Thioredoxin in Ecoli. and the fusion Proteincould been Purified quickly by ThioBond?resin. This was Key step to Produce CMIV Largely by fermentation. Meanwhile,the structure of the mutation II of CMIV showed a new way to design Peptide of cecropin which have the targeting sequence to tumor cell.