The Construction Of Non-Fusion Dissoluable Expression Vector Of Prokaryotic And Its Application

Exchanged the multiple cloning site (MCS)of pET32a into pET28a to form the plasmid pSYPU-O.Then clone the gene of protein H into pSYPU-0 at the site of Bpu11021 enzyme which is away from MCS to form the expression vector pSYPU-la. Used Analgesic-antitumor peptide(AGAP) as the expression model of pSYPU-la,constructed the recombine expression vector pSYPU-la-AGAP,transformed it into the E.coli BL21(DE3),and then induced the expression by adding IPTG,used SDS-PAGE to analyze the supernatant and precipitate,the protein had been expressed and the most of AGAP existed in the precipitate.Cloned the gene H into pSYPU-0 at the site of EcoR I and SacI,constructed the expression vector pSYPU-lb.Used AGAP as the expression model of pSYPU-lb,constructed the expression vector pSYPU-lb-AGAP ,with the same methods:IPTG induction and SDS-PAGE ,95% of the expression production existed in the supernatant.To test the stability of the expression vector pSYPU-lb,inoculated one time every 12h,after 116, 232, 348, 464 generation,the result was that the mutation rate of the pSYPU-lb is 0.003%, 0.013%, 0.017%, 0.021 %, the losing rate is 1.8 %, 5.0 %, 9.4 %, 17.23 % .It indicated that the expression vector have good stability.Transformed the recombined expression vector pSYPU-lb-AGAP into the expression host cell E.coli BL21(DE3), collected the host cell, lysed the host cell by sonication,collected the supernatant ,and used Folin method to measure the protein content,the content of the total...