Recombinant Expression And Activity Identification Of A Bioactive Peptide QUB-2995 From The Skin Secretion Of The Broad-folded Frog(Hylarana Latouchii)

Background:In recent years,the prevalence of diabetes mellitus in China has increased year by year.The latest epidemiological research shows that it is expected that 380 million people will be affected by the disease by 2025.Among the diabetic population,Type 2 diabetes mellitus(T2DM)is the most common type.Except for insulin resistance,β Cell dysfunction is the most critical pathogenesis of T2DM.Therefore,research and develop drugs with insulin releasing activity to stimulate β Cells secrete insulin and promote islets β Cell proliferation and regeneration has become the main direction of treatment of T2DM.At present,the clinical drugs for T2DM such as sulfonylurea,metformin and glinide have limited efficacy,tolerance and toxic and side effects.Therefore,it is very necessary to develop bioactive peptides that can regulate glucose metabolism and significantly reduce toxic and side effects.Bombesin polypeptide QUB2995 is a polypeptide with diversity bioactive functions isolated from the skin of Asian frog Hylarana latouchii,which has great research value.Objective:To find a novel bombesin polypeptide and study its biological activities.Methods:The skin secretion of Hylarana Latouchii was extracted by electrical stimulation;Single chain of bombesin polypeptide was cloned and sequenced by molecular cloning;QUB2995 was synthesized by solid phase synthesis;A synthetic peptide was purified by reverse phase high performance liquid chromatography;The synthetic peptide was confirmed by matrix assisted laser desorption time of flight mass spectrometry;qPCR and ELISA were used to detect the insulin secretion of MIN6 cells;Minimum inhibitory concentration was used to detect the antibacterial activity of QUB2995 against Escherichia coli,Staphylococcus aureus and Candida albicans;MTT assay was used to detect the anticancer effect of QUB2995 on human prostate cancer cell line PC-3;Hemolysis test was used to detect the toxicity of QUB2995 to red blood cells.Results:a novel bombesin polypeptide named QUB2995(GAFGDFLKGAALKILSIAQCKLSGTC)was found in the skin secretion of Hylarana latouchii.This peptide belongs to the Brevinin-2 family.It can significantly increase the insulin gene transcription level of MIN6 cells,promoting the insulin secretion of MIN6 cells.The most significant effect is at the concentration of 10-5 M.Besides,it can inhibit viabilities of Staphylococcus aureus,Escherichia coli and Candida albicans with MIC of 16 μM,16 μM and 64 μM,respectively.In addition,at a concentration of 1×10-4 μM,it can also kill prostate cancer cell lines.Its hemolysis is high and increases in a dose-dependent manner between 32 and 512 μM.