Research On The Expression Of Short-acting Human Insulin In Escherichia Coli

Diabetes is a kind of chronic metabolically disease has become a threat to human health. Insulin is the most effective first-line drugs to cure diabetes in clinic. The recombinant human insulin analogues were widely applied in clinic because they have a lot of advantages to treat diabetes. Especially, re-combinant human short-acting insulin analogues have advantages of the short pharmacodynamic peak time and quick working time. They also could im-prove the drug compliance for diabetic patients with insulin injections. The short-acting insulin analogues could also reduce the risk of the low blood sugar's occurrence during before next meal or the dawn. So short-acting insu-lin analogues were largely demanded due to many diabetes patients prefer them. However, there were few kinds of short-acting insulin analogues were used in clinic, such as insulin Aspart injection and Recombinant Lispro Insu-lin Injection. Therefore, it is necessary to develop new kinds and novel pro-duction ways for short-acting insulin analogues.In recent years, plant expression systems have advantages at cost; scale and safety to produce therapeutic proteins compare with traditional expres- sion systems such as Escherich coli, yeast and transgenic animals. Nowa-days, the plant expression system has become one of the most important medical protein expression systems.Considered above-mentioned reasons, based on the amino acid se-quences of human insulin (GenBank. AAA59172) and mini C-peptide (TYPGDVPK), the 198 bp gene Des 30 of coded short-acting human proin-sulin analogue was designed and synthesized according to codon usage bias of rape (Brassica napus L). Subsequently, the DNA sequences of the Des 30 was used as template, three genes of the B26H, B28D and B26H-B28D were created by PCR mutational technique, and four prokaryotic expression vec-tors of the pET28a-Des30, pET28a-B26H, pET28a-B28D and pET28a-B26H-B28D were constructed. The plasmids of the expression vec-tors were transformed into BL21(DE3)of the Escherichia coli. Target proteins were successfully induced by IPTG, isolated and purified by Ni-NTA bind resin, and target proteins were refold and digest, and then the bioactive were assayed for four insulin analogues. The research results were summarized as follows:1. Gene synthesis and constructs: Des30 gene was designed and synthesized according to rape codon usage preference. The 5'end and 3'end of the Des30 gene contained cutting sites of the BamH I and Sac I for cloning eas- ily. The series mutant insulin genes: B26H, B28D and B26H-B28D were created by PCR mutational technique. Thereafter, the target fragments were ligated into corresponding site of plasmid pET28a. The constructed pro-karyotic expression vectors of the pET28a-Des30,pET28a-B26H,pET28a-B28D and pET28a-B26H-B28D were intruded into competent cells of E.coli BL21 (DE3).2. The expression and purification of proinsulin analogues: Four proinsulin analogues were expressed in E.coli at inclusion body form by IPTG-induceing. The best induction time was confirmed through detected expression level of target protein at different induction time, and the results indicated that the best induction time was 6 h. The target proteins were puri-fied by the Ni-NTA resin.3. The refolding and digest of proinsulin analogues: In order to improve the refolded efficiency, the direction of tradition protein refolding buffer was optimized. The four proinsulin analogues were refolded at 4℃, and then were cleaved by trypsin and carboxypeptidase B. Western blot result implied that all four proinsulin analogues have immunocompetence. Results of the MALDI/TOF mass spectrometry showed that the mass of the cleaved pro-insulin analogues were identical with presumed human insulin.4. Bioactive assays: the data derived from mice blood sugar experiments demonstrated that the E.coil-derived short acting insulin analogues of the B26H- insulin,B28D-insulin and B26H-B28D-insulin could significantly reduce plasma glucose levels (P<0.05) compare with negative con-trols(non-injection or physiological saline) at a dose of 1U/Kg in courses, which do not have significant difference with NovoRapidR (Novo Nordisk Company), and the effect was found 20 min after injection to reduce plasma glucose, and reached peak at 120 min after injection. The effect of the E.coil-derived B28D-insulin was similar with human-insulin.This study could provide helpful information for study and design new kinds of efficiency insulin analogues, also provided important reference to express and recover of biologically active human insulin analogues using plant expression systems.