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Cloning Sequence Analysis And Expression In Prokaryotic And Activity Assay Of β-defensins Gene From Poultry

Posted on:2008-07-05Degree:MasterType:Thesis
Country:ChinaCandidate:J Z ShiFull Text:PDF
GTID:2120360218462052Subject:Animal breeding and genetics and breeding
Abstract/Summary:PDF Full Text Request
The experiment research Gallinacin-4 gene cloning,Escherichia coli expression and its bio-activity identification from poultry difference speciations.Defensins are imporce anti-bacterial peptides and anti-infect excitated specific exempt from conscription in animals and plants.Gallinacin-4(Gal-4)is importanceβ-defensin in vivo of birds.In order to acquire defensins peptides,experiment contruction gene procarryon ion carder and expression in Escherichia coli.The research refer to Genbank already deliver NM001001610 sequences, mature peptidc of NM001001610 sequences(CDS remove signal peptide and propiece)design primers,silly fowl,Gushi duck,Gushi goose,ostrich,dove extraction RNA form bone marrow,RT-PCR amplication about 117bp gene fragments,and clone pUCm-T Vector,establish pUCm-Gal-4 of recombinant plasmid, PCR,the enzyme cut identification and DNA gene sequence identification,This gene Gal-4 is code sequence,comparative indicate:five breeds Gal-4 gene sequences and deduction amino acid sequences all are 117bp,code 38 amino acid sequences, nucleotide homology arrive at 100%:AY621319 reportorial Gal-4 gene sequence complete accordance.NM001001610 sequence have only one base differece.(Gushi duck has one base mutation,but coded amino acid sequence no change.There were three differences of cause analyse:1.there might be speciations among themselves exist such mutation:2.PCR inflict bases mutation likelhood:3.there might be gene sequence exist error and so forth.)record registration Genbank,The accession number was:silky fowl DQ673442,dove DQ860106,ostrich EF031035,Gushi duck EF606702,Gushi goose EF606703.We get soluble expression products ofβ—defensins,which provides a sound basis for future research on its activity and purification..To lay the foundation of furher cloning,expressing the Gal-4 gene, further studying of the function of the Gal-4 gene prodct. Analysis of Gal-4 maturation peptides DNA sequence 114bp(contain one ending codons),five Rare Codons by Substitution of prokaryotic common. Codons.Afresh compound 114bp gene,amino acid sequence(38 entries)still amino acid sequence originally.Adhibit BamHI and SalIrestriction sites respectivel.Subclone similarly,using BamHI and SalI to double-cut p GEX-4T-lexpression vector and then link these two double-cut products together and transfer.Recombinant plasmid pGEX-Gal-4 transformed host strain BL21(DE3)PlySs,The expression was induced by IPTG.,SDS-PAGE electrophoretic display,pGEX-Gal-4 expressed protein was approximately 30KDa in molecular weight.,Results:The measured relative molecular mass was accorded with the theoretical value,The results of study on inducing and thecondition for Gal-4 protein expression was optimized. 1.0mmol/L,37℃,4 hours.The cloning and expression of the Gal-4 gene established the foundation of the further research about the function and application of the Gal-4 gene.This study lays a foundation for further research.The crude extricated substance of pGEX-4T-1-Gal fusion protein no inhibitory effect on Staphylococcus aureus,Escierichia Coli,funguses(Candida albicans)by agar diffusion test.
Keywords/Search Tags:Poulty, β—defensins, cloning, pGEX-4T-1, expression
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