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Cloning And Prokaryotic Expression Of N/OFO Gene Of Mouse

Posted on:2014-08-24Degree:MasterType:Thesis
Country:ChinaCandidate:G Z LiuFull Text:PDF
GTID:2250330425952795Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
As a kind of endogenous opioid peptide receptors,N/OFQ belongs to the Gprotein coupled receptors.And it is widely distributed in the central and peripheraltissue.The higher expression of the gene in the whole area of the brain suggests that itmay have corresponding regulation function in the process of feeding and energymetabolism.And the neuroanatomy orientation of the gene is consistent with theenergy balance related area.In order to study biological functions of the protein codedby N/OFQ gene in mice further,this experiment was carried out the followingoperations:First,the test use the kunming mice as research objects,designed primersaccording to published gene sequences of N/OFQ in NCBI,and cloned N/OFQ geneusing the method of RT-PCR.And then constructed its prokaryotic expression vectorof pET-28-N/OFQ,after that induced its protein expression by IPTG(1.0mmol/L).Atlast,detected the protein by SDS-PAGE and Western blotting.The main results were asfollows:1, Cloning and Bioinformatics analysis of N/OFQ Gene of miceExtracted total RNA from brain tissue of mouse and cloned N/OFQ gene usedthe method of RT-PCR, The gene contains a complete ORF of564bp, encoding187amino acids, wherein the acidic amino acids39,basic amino acids29.And themolecular weight of the protein is21.71kDa, its isoelectric point is8.70. Afteranalysed by software,it founded that nucleotide homology are92.8%and85.2%compared with rat and martes, and the amino acid sequence homology are98.3%and87.5%compared with Ovis and Bos,it is higher than others.Phylogenetic analysisshows that the genetic relationship of mouse is closest to rat, and they are on the samebrancha,but it is farther than others.2,Prokaryotic expression of N/OFQ gene of miceIn order to obtain the N/OFQ protein molecules, this test designs synthesis of apair of expression with primer based on the cloning sequence.The N/OFQ sequence ofmice is inserted into the pET in the pET-28a (+).Identified by PCR of bacteria liquid,double enzyme digestion and sequencing identification,and it confirmed that theprokaryotic expression vector of N/OFQ gene builded successfully,and named itpET-28-N/OFQ. After that,the recombinant plasmid of pET-28-N/OFQ is transformed into E.coliRosetta.And then, the protein is induced to express in the condition of30℃,theconcentration of IPTG is1.0mmol/L,and the time of induction is6hours.Afterprocessing the product, it is identified by ways of SDS-PAGE and Westernblotting.And it is purified by the6×His Ni-NTA protein purification column.Theresult of SDS-PAGE shows that a single strip with the relative molecular weight isabout20kDa,and it conforms to anticipation.And it shows that fusion protein isexpressed in E.coli.At last,the purified protein was detected by Western blottingshowed that the protein obtained is the purpose of protein N/OFQ.This study have cloned the N/OFQ gene of mice, and structured its prokaryoticexpression vector successfully, got the target protein by prokaryotic expression.Andthe results of the study provides a basis for further study the biological function ofN/OFQ gene in mice.
Keywords/Search Tags:Mouse, N/OFQ, Cloning, Sequence analysis, Prokaryotic expression
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