Cloning And Expression Of The Key Enzymic Genes For Synthesis Of Guanine Arabinoside

Nucleosides and derivants are the main sources of anticancer and antiviral medicines.Guanine arabinoside(ara-G),an active metabolite of nelarabine,is also a key anticancer nucleoside analogues to inhibit the cell activity of T cell leukemia in a purposeful way as a prodrug of nelarabine etc.The chemical synthesis of ara-G is extremely complex and resulting in serious harmfulness to the environment and human for large amounts of chemical reagents used.Although biocatalysis has solved these problems to some extent,there are also a low amount of enzyme in the wild strain,and the bacteria itself has pathogenicity,which seriously hinders the industrial production.Therefore,in this paper,molecular biology technology were used to clone and express the key enzymes used in the biosynthesis of ara-G synthesis.Ggenetical engineered bacteria with high enzyme activity and high nucleoside transformation ability were obtained to laid foundation for large-scale industrialized production.1.Engineering bacteria construction of the purine nucleoside phosphorylase gene(deo D)and the uridine phosphorylase gene(udp):According to the sequences of genes deo D and udp in GenBank,primers were designed to amplify the deoD and udp genes from the Escherichia coli AEM0812 strain by PCR.The gene fragments were respectively ligated with the cloning vector pMD18-T and the recombinant plasmids pMD18-T-deoD and pMD18-T-udp obtained.Then recombinant plasmids were introduced into E.coli DH5?,and the clone strains with target genes were obtained.They were then ligated into the expression vector pET-28 a to construct the recombinant plasmids pET-28a-deoD and pET-28a-udp,then introduced into E.coli BL21(DE3)obtained the engineering strains E.coli(deoD)and E.coli(udp).Induced by IPTG,SDS-PAGE showed that the target gene were successfully expressed.The enzyme production capacity of the engineering strains E.coli(deoD)and E.coli(udp)were respectively 1.9 times and 1.2 times that of the original strain.2.Synthesis of arabinose 2,6-diamino purine nucleoside(ara-DA)catalyzed by E.coli(deoD)or E.coli(udp):The free cells of E.coli(deoD)or E.coli(udp)or the crude enzyme prepared with the stains were used to catalyze the first reaction espectively,and ara-DA was generated sucessfully.The free cells transformation rate of E.coli(deoD),E.coli(udp)were increased by 24.6% or 22.3% respectively.The crude enzyme solution were increased by 28.2%,2.0% respectively,compared with the original strain AEM0812.3.Construction of the adenosine deaminase gene(add)engineering bacteria:The add gene from Pseudomonas aeruginosa TX16 strain was cloned and then introduced into E.coli BL21(DE3)to obtain the engineered strain E.coli(add)with the recombinant plasmid pET-28a-add.After induced by IPTG,SDS-PAGE showed that the target gene was successfully expressed.The enzyme activity of the crude enzyme solution of E.coli(add)was 2.9 times that of the original strain P.aeruginosa TX16.4.Ara-G synthesis catalyzed by E.coli(add):The E.coli(add)free cells were directly added to the second step reaction system,HPLC results showed that ara-G could hardly synthesized using E.coli(add)free cells as catalyzer.The crude enzyme prepared of E.coli(add)and then reacted.But the reaction was successfully catalyzed with the crude enzyme prepared of E.coli(add).The conversion rate 96.8% was significantly higher than that of the original strain P.aeruginosa TX16,which was 2.2 times that of the original strain.