Expression Of Gene Of The Fibrinolytic Enzyme In Escherichia Coli And Pichia Pastor

Thrombotic disease has become a common and frequently-occurring disease, it is listed the first in disability and mortality and seriously affected the life quality and span of human. For the requirements of clinical application of thrombolytic agents, research and development of specific, efficient, safe, inexpensive thrombolytic agents have broad application prospects. By using of molecular biology and genetic engineering techniques and the advantages of existing thrombolytic agents, it is a research focus on designing the perfect thrombolytic agents at home and abroad. At the same time, it will be the future research directions to find the new natural sources of thrombolytic agents and explore new structure thrombolytic agents.Fibrinolytic enzyme BpFE from B. pseudomycoides is a new enzyme isolated from soil. Gene length 1701 bp (GenBank FJ463037), which is 954 bp mature peptide. It was reported front-end sequences of mature peptide (gene length 747 bp) may be related to the regulation. In order to further explore the relationship between the full peptide and the mature peptide, the role of front-end sequences and the effect of activity, so we do the work of mature peptide gene cloning and expression. For future production and research we explored expression in Pichia pastoris, the main contents are as follows:Designed and synthesized a pair of primers according to DNA sequence(GenBank FJ463037) of B. pseudomycoides fibrinolytic enzyme. We also added EcoRI and XhoI restriction site on both ends. Mature peptide G of plasminogen was amplified. The purified vector pET-28a and PCR product of mature peptide gene are digested by EcoRI and XhoI. After purifying the digested products respectively, we ligated these two kinds of DNA by T4 DNA ligase and constructed the recombinant plasmid pET-28a-G. It was sequenced after the recombinant plasmid of pET-28a-G was transformed into host bacterium of DH5α. The sequencing results showed that the insertion sequence length 954bp, encoding 317 amino acids, and the mature peptide 954bp sequence was the same with the mature peptide of GenBank. Neutral zinc metallopeptidases, zinc-binding region signature (sequence VIGHELTHAV) has not changed.The recombinant plasmid of pET-28a-G was transformed into host bacterium of E.coli BL21 to construct pET-28a-G/BL21 engineering strains successfully. SDS-PAGE analysis was performed after pET-28a-G/BL21 being induced by IPTG(1 mmol·L-1) to detect intracellular expression of the fusion protein. Fusion protein had an apparent molecular weight of about 40kD, which was consistent with the predicted results. Fibrin-Plate method showed that the intracellular protein had activity, and nickel affinity chromatography purified products which it had activity. This shows that front-end sequences of mature peptide sequences may be related to the regulation and had no direct effect on the fibrinolytic enzyme activity, so the part of the mature peptide had fibrinolytic enzyme activity.In order to gain extracellular expression and lead inconvenience to production, we cloned of the full gene encoding fibrinolytic enzyme BpFE and expression in Pichia pastoris GS115 for research preliminary. we designed and synthesized a pair of primers according to DNA sequence(GenBank FJ463037) of B. pseudomycoides fibrinolytic enzyme and polyclonal sites of eukaryotic expression vector pPIC9K to construct pPIC9K-G vector. We also added EcoRI and NotI restriction site on both ends. Recombinant plasmids were transformed to Pichia pastoris GS115 after being linearized. After MD MM and G418 pressure screening, positive transformation strains were obtained. After being induced at 25℃in methanol. Protein had an apparent molecular weight of about 64kD and it showed the fibrinolytic enzyme activity. After nickel affinity chromatography purified fibrin-plate method showed that the products had activity.By using molecular clone and gene reconstruction technology, we not only successfully expressed the mature peptide gene to discuss the relationship the full length and the mature peptid gene of fibrinolytic enzyme BpFE, but also initially detected effective expression of full-length gene of fibrinolytic enzyme BpFE in Pichia pastoris. Our experiment will lay a necessary theory and practical foundation for the development of genetic engineering drugs.