Construction Of A Streptomyces Expression Vector And Cloning Of A Large Fragment Of A Doxorubicin Biosynthesis Gene Cluster

Many peptides or proteins of basic research and applied value is very low in the natural biological.So there are many restrictions in the further study of the nature of these substances,development and application.Application of genetic engineering technology,these peptides and proteins gene can effectively be cloned into a variety of gene expression vector,resulting over expression in such as Escherichia coli,Bacillus subtilis,Streptomyces,yeast,animal and plant species.In recent years,DNA recombination,gene clone,fixed point mutations in vitro,the base sequence analysis has been widely used in molecular biology technologies such as Streptomyces genetics research,Streptomyces vector,phage carrier,and receptor system of Streptomyces have very big development,Suitable for Streptomyces conversion technology has been very mature,All these have created favorable conditions for the further study of Streptomyces.Based on the above conditions,This study constructed the Streptomyces vector contain e.coli replicator(pIJ101 ori),strong promoter(PtipA)and histidine tag(6 xhis tag),TEV protease and the expression vector don’t containing histidine label.Replicators of e.coli in the cloning of the gene in e.coli expression;Sulfur chain wire rhzomorph resistance genes(TSR)to filter the correct clone;the strong promoter(PtipA),it has the strict closure and openness,has become the most valuable in the construction of expression vector Streptomyces one of the promoter.These two kinds of Streptomyces expression vector,containing histidine tag(6 xhis tag)and TEV proteases cut site expression vector,can through the Ni-NTA protein isolation and purification of affinity chromatography,can also be expressed through TEV enzyme loci enzyme isolated protein.expression vector that does not contain histidine labels and TEV protease cutting locus to provide more choices for subsequent protein expression,is advantageous to the gene cloning.Adriamycin has an effect on a wide variety of tumor,is cycle non-specific drug,have all sorts of growth cycle of tumor cells to kill,its antineoplastic spectrum is wide,and can produce a wide range of biological chemistry,it is now one of the most widely used clinically antitumor drugs.The adriamycin mainly through chemical semi-synthetic method synthesis.typically by soft erythromycin after complex chemical synthesis and get it.Using this method synthesis doxorubicin has many advantages,but also a lot of deficiencies,for example,consume large amounts of organic solvents,environmental pollution,yield is generally low.So imagine build microbial expression vector,heterologous expression of direct production of adriamycin.This research mainly by PCR technology from Streptomyces genome amplification required purpose gene fragments,And step by step to the PCR amplified out small fragments of gene splicing,become the purpose gene fragment,In the end,joining together into a complete expression vector.Although this program is trival,complex,and spend a certain amount of time and manpower and material resources,but it can reduce cost,simplify the production process,reduce pollution,and bring certain social benefits and economic benefits.