Study On The Production And Activity Of A New Type Of Insulin Analogues

Objective: Type Ⅰ diabetes mellitus(TIDM)is a metabolic disease characterized by permanent destruction of beta cells.To maintain glucose homeostasis,TIDM patients need injection of insulin throughout their lives.Frequent injection of insulin not only causes discomfort to patients,but also significantly increase the risk of hypoglycemia.Transferrin(Tf)has been used by many researchers in production of recombinant protein.Proinsulin-Tf is a fusion protein that has been produced in HEK293 cells and was shown to been long-term effective and liver specific.In order to increase production,in this study,we use Escherichia coli to induce the expression of ProINS-Tf and to detect its activity.In order to further improve the yield,we used molecular biological methods to reverse the C-terminal sequence of transferrin and express and purify it.The yield and activity of the two recombinant proteins were compared so as to lay a foundation for the industrial production of a new type of insulin analogue.Methods: The recombinant plasmid pET28a/Pro INS-Tf was designed and synthesized,and the primer was designed to obtain the sequence of ProINS-NTf by PCR.The recombinant plasmid pET28a/ProINS-NTf was digested into pET28 a expression vector to express ProINS-NTf.The two plasmids were transformed into BL21(DE3),and the recombinant protein was induced by IPTG.The recombinant protein was purified by nickel column affinity chromatography and its activity was detected by injecting purified recombinant protein into streptozotocin(STZ)-induced type I diabetic mice.Results: The two recombinant plasmids were transformed into BL21(DE3)and the target proteins with molecular weight of 90 kDa and 50 kDa were induced and expressed respectively.The recombinant proteins in the form of Pro INS-Tf and ProINS-NTf were transformed into soluble form after denaturation and renaturation.The purified recombinant protein was purified by nickel column affinity chromatography,and the purified ProINS-Tf(67.5 nmol/kg)and ProINS-NTf were subcutaneous injected into STZ-induced type I diabetic mice.In the Pro INS-Tf(67.5 nmol/kg)group,the blood glucose decreased slowly and maintained at a low level for a long time.The blood glucose of the Pro INS-NTf(67.5 nmol/kg)group did not decrease significantly.Conclusion: 1: ProINS-Tf can be highly expressed by pET28a-Pro INS-Tf/BL21,a highly purified and biologically active target protein was obtained after denaturation,renaturation and purification.2: ProINS-NTf does not have the same hypoglycemic activity as ProINS-Tf.