Cloning And Prokaryotic Expression Construction And The Eukaryotic Expression Vector Of Duolang Sheep IL-1β In Xinjiang

Interleukin-1β （IL-1β） is produced by macrophages, has biological activitiescomplex. IL-1β is an important cytokine in inflammatory reaction of the body. when in lowconcentration in partially time, with immune regulation function. To stimulate antigenpresenting cell together and T cell activation. To being promote B cell proliferation andantibody secretion. When the body produces a large number of IL-1beta will produceendocrine effects: induced synthesisand cause fever and cachexia hepatic acute phase protein.This experiment design according to NM₀01009465.2of gene sequence specific primerin GenBank, using Xinjiang Duolang sheep lymphocytes of total RNA as atemplate byRT-PCR Duo-lang sheep IL-1β gene sequences. Connected to the pMD-18T carrier and usedthe method of heat shock into host bacterium DH5α, which after gain positive clones ofenzyme digestion and bacteria liquid PCR identification, and then sequenced. Through thenucleotide and amino acid sequence alignment occurrence that IL-1βgene sequencecontains a862bp open reading frame and mature protein encoded of amino acidsequence length is286amino acids. Through GenBank in Blastn sequence analysis,Duolang sheep IL-1β gene homology with ordinary sheep as high as99%.With cattle,antelope, dolphins, wild boar compares,its has96%,96%,82%,86%respectively. IL-1βg e ne co ntain s p otential O-g l ycosylation s ite s, o ne N-gl yc o s yl a t ion s it e s, te nphospho-rylationsites.The recombinant prokaryotic expression vector of pET-28b-IL-1βXinjiang Duo liang was constructed successfully. After induction, fusion protein wasexpressed and its expression the molecular weight of32.4KD. The6×His tag of fusionprotein has a good respon to the original by wester blot occurrence.By the animal disease prevention and control laboratory-80℃kept pMD-18T-IL-1βbacteria liquid after activation, the extraction of plasmid double enzyme, and pET-28bprokaryotic expression vector connection, transformation into the E.coli DE3host bacteriaof screening positive clones and obtain monoclonal. Positive clones of gene wereidentifiedby PCR and restriction enzyme digestion and then sequenced. With a final concentration of1mmol/L IPTG induction, the IL-1β protein expression in Duo Liang weredetected by electrophoresis SDS-PAGE and check if western blot test is for the purposeof protein.pMD-18T-IL-1β bacteria liquid preserved by-80℃.Through the activation of extraction ofplasmid by enzyme digestion, and connect with eukaryotic expression plasmid pcDNA3.1and transformed into host strain DH5α. To be screening positive monoclonal after bacterialiquid PCR and double enzyme digestion and sequencing. The successful construct of XinjiangDuolang sheep IL-1β-pcDNA3.1Eukaryotic Expression plasmid.