Mining,Optimizing And Functional Verification Of Calcitonin Genes From The Marine Genome Database

Backgrounds:China has gradually entered the aging society,osteoporosis is a common disease in the elderly,especially middle-aged and elderly women.Although calcitonin is an effective drug used in clinical to treat osteoporosis,it also has many problems such as high cost,short half-life,high immunogenicity problems.Therefore,to explore more efficient calcitonin has important clinical significance.Objects:Through the sequence comparison,phylogenetic tree analysis and activity detection of many new types of calcitonin(CT)gene sequences of marine origin,the discovery of highly active new CT laid the foundation for clinical application;and established the technology platform of high-throughput excavation to verify the active polypeptide,in order to discover the new activity,the new function polypeptide provides the technical reserve.Content:1.Using bioinformatics methods to mine the CT sequences of different species from the marine organism genome database.2.Analysis of CT sequence differences,chemical synthesis,mass spectrometry analysis and activity verification.3.Summarize the activity rules,and based on this,design and optimizing of new CT sequence and functional verification.4.A preliminary screening platform for marine biologically active peptides was established through the study of calcitonin.Methods:1.The CT sequences of other species were analyzed and obtained by BLAST tool via the salmon calcitonin(s CT)sequence as the query sequence,consisting of two parts: NCBI non-redundant protein library and BGI gene aquatic organism genome database.2.Sequence comparison using clustal omega software,combining species-selective representative sequences to construct evolutionary tree using MEGA7 software.3.According to the evolutionary tree results and species classification,13 sequences including s CT were selected for chemical synthesis.4.In accordance with the requirements of Chinese Pharmacopoeia,the biological activities of CTs were estimated using the standard of the rat hypocalcemic activity assay in vivo.5.CD spectrum analysis was performed on 13 sequences including salmon calcitonin,and various physicochemical properties of each calcitonin sequence were calculated using the Prot Param tool,such as isoelectric point,GRAVY value,estimated half-life,etc.;using Heli Quest The amino acid composition ratio,hydrophobicity,etc.of each calcitonin sequence were analyzed.The spatial structure of each calcitonin sequence was evaluated and predicted using software such as Swiss-Model,Swiss-Pdb Viewer,and PEP-FOLD.6.Based on the results of calcitonin activity,sequence and spatial characteristics,summarize the law of activity.And based on this,computer-aided design optimizes the new calcitonin sequence.7.Design CT sequences for chemical synthesis.8.In accordance with the requirements of Chinese Pharmacopoeia,the biological activities of designed CTs were estimated using the standard of the rat hypocalcemic activity assay in vivo.9.The half-life of designed CT sequences were determined by ELISA.10.The immunogenicity of designed CT sequences were determined by ELISA.Results:1.A total of 64 candidate CT gene and amino acid sequences from different species were obtained by BLAST using the s CT sequence as the query sequence,such as the cartilaginous fish class,fin fish class,amphibia,lizard(bird class),Achilles(Mammalia)class.2.Select 26 species representative CT sequences to construct the evolutionary tree.3.Based on the characteristics of biological evolution,13 sequences including s CT and human calcitonin(h CT)were selected for chemical synthesis.4.The activity of 13 CT sequences were validated in rats,of which four types of candidate CTs had 30% higher activities than s CT,three other types had similar activities to s CT,and the remaining four types had much lower activities than s CT.5.CD Spectral analysis showed that these 11 natural calcitonin secondary structures are similar to salmon calcitonin.6.Software analysis showed that the higher activity of the calcitonin sequence usually has a higher hydrophilicity.This may be related to the overall "flexibility" of CT.7.Sequence comparison analysis showed that the presence of phenylalanine(Phe)/isoleucine(Ile)at position 16 or 22 significantly affected the activity of CT sequence.This may affect the stability of the alpha helix and the folding of the C-terminus.8.Based on the above results,6 CT sequences were designed artificially.9.The activity of 6 newly designed CT sequences(CT-01~06)were validated in rats,of which 2 sequences had 50% higher activities than s CT,1 sequence had 30% higher activities than s CT,and the remaining 3 were lower than salmon calcitonin.10.The half-life of the 3 sequences of CT-01,CT-04 and CT-06 in rats was analyzed by ELISA method,which was found to be comparable to s CT.11.The immunogenicity of the 3 sequences of CT-01,CT-04 and CT-06 in mice animal models were analyzed by ELISA method,which was found to be lower than s CT.Conclusions:1.By detecting the activity of calcitonin in natural marine organisms,it was found that the activities of Mdo,Oan,Gga,and Cau were 30% higher than that of salmon calcitonin,and the activities of Tni,Pbi,and Loc were comparable to those of salmon calcitonin.Cmi,Spa,Bpe and Hip 4 calcitonins were lower than salmon calcitonin.2.By summarizing the laws of calcitonin activity,three high activities,long half-life,low immunogenicity of calcitonin sequences were finally designed and optimized.3.The research group has established a high-throughput technology platform for excavating and verifying active peptides through the study of calcitonin.