Production And Characterization Of Human Recombinant Tumor Endothelial Marker 8 (TEM8) SV3 Variant

In this investigation,according to the data of 96%amino acid homology between the human and mouse proteins and the original studies on the mice recombinant tumor endothelial marker 8(TEM8) SV3 variant,the human recombinant tumor endothelial marker 8(TEM8) SV3 variant were proposed to construct and characterize its property.This research to be done in order to acquire an antigen,which would be used as an immunogen to immunize mice.In this study,by using the template of human breast tumor cell lines endothelial marker 8(TEM8)SV3 DNA,we designed the primers and then the DNA was amplified.The cloning vector was constructed by the GATEWAY technology by two different steps:LR reaction——entering clone and BP reaction——expressing clone,obtaining the recombinant DNA we needed.In the clone expression study,two different competent cells were used:E coli.DH5αand BL21 Star(TM)(DE3).The results of expression in different competent cells were analyzed and the expression condition was optimized.The recombinant DNA acquired in the last step was sequenced,optimized and the highest homology product was collected,then it was induced to protein expression.In the experiment of protein induction,The induced time(3 hours and 5 hours),induced reagents(NaCl and IPTG) and the competent cells(E coli.DH5αand BL21 Star(TM)(DE3)) were optimized,respectively.The product was collected and the protein was extracted.At last,the product was preliminary purified and done the Western blotting by using the commercial TEM8 antibody.The results were as follows:1.We successfully acquired human TEM8 SV3 DNA with the molecular weight of 1000bp through the GATEWAY clone technique.2.The competent cells played a great role not only in the clone system but also in the expression system.In the clone process,the competent cell E.coli DH5αdid not bear the gene for T7 RNA polymerase and maintained the construction condition within it.All these reasons resulted in the lower concentration of DNA and the RNA contamination of DNA.The problem also emerged in the protein induction.That resulted in the failed protein expression.3.In our induction study,we acquired the suitable expression condition below:The induced time:3 hours;The induced reagent:IPTG and the competent cells:BL21 Star(TM)(DE3).4.We got an expression protein in the lysate of the clone with the molecular weight of 43KD,which was similar to the previous data of mouse recombinant TEM8 SV3 protein with the molecular weight of 38KD. We preliminary predicted this protein as the human recombinant tumor endothelial marker 8(TEM8) SV3 variant.The results of the preliminary purified and western blotting were confirmed that the recombinant protein had the specific antigen characteristics of the human recombinant tumor endothelial marker 8(TEM8) SV3 variant.