Clone Of Streptomycete MY0504 Plasmin Gene YG4 And Expression In Escherichia Coli

The formation of arterial and venous thrombosis has caused approximately 18 million deaths all over the world each year.Therefore,the treatment of thrombosis is a medical and health problem that people around the world care about.The most effective and reliable way to treat this disease is to use drugs.At present,antiplatelet and anticoagulants drugs are the most commonly used drugs in clinical practice.However,there are few studies on direct thrombolytic drugs.Moreover,the thrombotic drugs used currently for prevention and treatment in clinic have some disadvantages,such as high risk of bleeding,short half-life and high cost.Therefore,it is particularly urgent to develop new thrombolytic drugs that can make up for these defects and are more suitable for human.The special growth environment of marine actinomycetes determines that their physiological characteristics and metabolic pathways are different from those of terrestrial organisms.It is of great practical significance to study their metabolites.Marine actinomycetes have become an important resource for discovering new bioactive substances,which open up a new direction for the search for new thrombolytic drugs.In this paper,Streptomycetesp.MY0504,which is an actinomycete secreting fibrinolytic enzymes and was isolated from the ocean in the previous works by our laboratory,was sequenced by Pac Bio RS II.After splicing,the whole genome sequence of Streptomycetesp.MY0504 was obtained.The genome size was 7,134,390 bp and the GC content was 73.31%.In the gene,there were 6137 functional genes that account for 87.74% of the genome,the total length of which was 6,238,047 bp and the average length of which was 1016 bp.And there were 22 gene islands,3 prehages and 80 CRISPR sequences in the genome,which was inferred.By gene function annotation,four genes which were assumed to have serine protease were screened out.The plasmin YG4 gene in Marine Streptomyces was cloned and was analyzed by bioinformatics methods.The results showed that the size of YG4 gene was 1083 bp and that encoded 360 amino acids which formed a stable hydrophilic protein that was without signal peptides and transmembrane domain.The advanced structure of the protein was dominated by random coil.The rich advanced structure played an important role in the protein biological function.The recombinant bacteria p ET-30a(+)-YG4/BL21(DE3)was constructed and induced with IPTG.Recombinant proteins were detected by Tricine-PGAE and Western-blotting.The results showed that an expected protein band appeared at 38 k Da in the test group inducted,which was specifically bound by His-tag mouse monoclonal antibody,and that the recombinant YG4 protein was successfully expressed.