Cloning Expression And Enzyme Activity Analysis Of The Serpin23 In Aedes Aegypti

Objective:This study aimed to clone and express Serpin23recombinant protein of Aedes aegypti from Guangdong Dianbai strain in the prokaryotic,wheat germ cell-free and mamma Lian cell expression,for exploring its possible biological functions.Methods:Specific primers were designed by the Serpin23 gene,and we used the PCR technology to amplify the Serpin23 gene in a mature peptide,cloning the sequence into the pGEX-4T-1 plasmid and the vector,pEU-E01-His-TEU-MCS-N2,from the wheat germ cell-free expression.The recombinant plasmid pGEX-4T-1-Serpin23（rSerpin23）was further transformed into BL21（DE3）,an expression fungus,and induced by IPTG.The recombinant plasmid pEU-serpin23（rpEU-serpin23）was expressed according to the instruction of wheat germ cell-free expression kit.The recombinant plasmid pcDNA3.1-serpin23 was constructed and transfected to the 293T cell,and the cells and the supernatant were collected after the culture on DMEM for 48h.The expression products were identified by SDS-PAGE and Western blotting.The identified proteins were purified by GSTrap^TMFF and Ni-NTA affinity chromatography column.The manual method for the detection of rAa-Yc on human whole blood prothrombin time（PT）,thrombin time（TT）,and activated partial thromboplastin time（APTT）.The inhibitory effect of rSerpin23,rpEU-Serpin23 and pcDNA3.1-Serpin23 on trypsin was detected by chromogenic substrate.Results:The full-length of Serpin23 was 1254 bp with a signal peptide,and the bioinformatic results showed that it had a domain of Serpin structure.PCR amplification obtained its mature peptide sequence and it was cloned into the vector of PGEX-4T-1 and pEU-E01-His-TEU-MCS-N2.The results of PCR,restriction enzyme identification and DNA sequencing demonstrated that rSerpin23recombinant plasmid was successfully constructed.The 1197 bp of the mature peptide encoded 399 amino acids.We found a synonymous mutation of T（thymine）changing to C（Cytosine）at position 904 compared with the sequence of NCBI（Gen Bank Accession:DQ439995）.Then a pGEX-4T-1-Serpin23 prokaryotic expression vector was constructed and transformed into BL21（DE3）.A soluble protein of rSerpin23 was obtained by IPTG inducing.The recombinant plasmid pEU-Serpin23was successfully expressed as the recombinant protein（rpEU-Serpin23）using the wheat germ cell-free expressional system.Recombinant plasmid pcDNA3.1-Serpin23was transfected into the 293T cell and detected with the recombinant protein expression in the cell.The recombinant protein rSerpin23,rpEU-Serpin23 and pcDNA3.1-Serpin23 were identified by Western blotting as the target protein.The PT,TT and APTT were 13.29 seconds,15.34 seconds and 36.72 seconds respectively when 1.0μmol rSerpin23 protein was mixed with the human whole blood.In addition,the optical density of the enzyme dynamic detection indicated that the recombinant protein of rSerpin23,rpEU-Serpin2 and pcDNA3.1-serpin23 had no inhibiting trypsin activity.Conclusion:Serpin23 recombinant protein of Aedes aegypti from Guangdong Dianbai strain was obtained successfully with all of the three methods,and the inhibitory activity of trypsin was not detected.