| Glucagon-like peptide-1(GLP-1) secreted from gut L cells, which has two activity forms. One is a full length of GLP-1(7-37), another is GLP-1(7-36)-NH2. GLP-1 can stimulate the beta pancreas islet to secrete insulin in hyperglycemia, meanwhile prevent glucagon secretion, promote the pancreatic beta cells proliferation, induce stem cells to differentiate into islet cells, reduce gastric emptying. Therefore GLP-1 become a potential drugs for the treatment of type 2 diabetes. Preliminary results of our lab showed that the activity of two GLP-1 connected at the HSA N-terminal was not very high, meanwhile the N-terminal AA of GGH was not consistency. Base on this results, we studied the fusion durg to improve the activity and keep N-terminal stability in this paper. The main results are as follows:(1) To provide a simple and reliable method to screening GLP-1 analogue, a cell modle to detect GLP-1 analogue activity was constructed. One cell(CHO/pcDNA3.1/hGLP-1R) was finally selected by monoclonal selection and genetic toxin(G418) pressure selection. RT-PCR, flow cytometry and laser confocal results showed that the cell model was successful constructed. Activity test results showed that the cell model could be used in the detection of GLP-1 analogues activity, and the response value cell model was high than wild strains(RINm5f). There by, five GLP-1 analogues was designed, and obtained by chemical synthesis. The in vitro activity results showed that effective drug concentration increased over 23 times than positive control group. The properties was not strong for GLP-1 mutant to become drugs.(2) P.P astoris/pPIC9K/GGH was constructed, which retained Kex2 and Ste13 two signal peptide enzymes to express fusion protein of AOX/GGH. In P.Pastoris expression system, the drug protein was designed and the results showed as follows. 1. To eliminate the side effects of methanol on the yeast, the GGH expressed in P.Pastoris useing glyceraldehyde 3 phosphate dehydrogenase(GAP) promoter. Results showed that the expression and activity of two production was the same, but the fermentation time with GAP promoter was less 20 h than AOX promoter, and the fermentation process was more easy and convenient. 2. To eliminate the side effects of Ste13 signal peptide, we removed signal peptide enzyme Ste13 and only keep Kex2 signal peptide enzyme to express GGH. The activity of drug protein was increased by 1.45 times, but the fermentation yield decrease by 31%. 3. To obtain the best serial number of GLP-1 in fusion protein and enhance consistency of N-terminal in fusion protein, We added His-tag with GLP-1 and HSA fusion protein, meanwhile adding an enterokinase(EK) at the connection. Then, we design single double and triplets GLP-1(A8G) respectively connected with the N-terminal of HSA. What more, one AA(ala) was added to each fusion protein at the N-terminal. Results showed that two GLP-1 conjoined with HSA could reduce the space resistance between HSA with GLP-1, and the activity could improve. 9K/NGGH was obtained by using the his-tag and EK. The final purification recovery increased 50% and the activity increased 4.66 times compared with AOX/GGH. What more, N-terminal of 9K/NGGH was more consistency than AOX/GGH.(3) To obtain more high activity of GGH protian, recombinant CHO cells was constructed to express GGH protein. Three recombinant CHO cells(CHO/pMH3/GGH, CHO/pMH3/AGGH, CHO/pMH3/NGGH) were finally constructed after four cycles screening. Verified with WB and cell stability, cells were fermentation in 3 L flask. The protein yield of CHO/pMH3/GGH was 437 mg/L after fermentation 16 days, CHO/pMH3/AGGH was 358 mg/L, and CHO/pMH3/NGGH was 322 mg/L. The yield in 3 L fermentation was six times higher than flask. The highest activity in CHO cells was CHO/NGGH, and the activity was 1.36 times higher than 9K/NGGH.(4) To detect the new drug substance quality of CHO/NGGH compared with “2015 China pharmacopoeiaâ€. Test results show that the final CHO/NGGH meet the general requirements of pharmacopoeia. Pharmacological of CHO/NGGH was further researched. CHO/NGGH could effectively stimulate proliferation of RINm5 f cells at 100 nM. Cells density could increased about 0.5 times after three days. Drug could differentiate MSC to cells cluster after 5 day with 500 ng/mL(6.9 nM) CHO/NGGH. The cells cluster was preliminary proved to pancreas cells by DTZ dyeing. The cells cluster could transcribe the marker gene of pancreas cells, what more, the cells cluster could secret insulin. All results showed that the cells cluster was the pancreas cells. |
