牦牛Prdm9基因保守区的原核表达及多克隆抗体制备
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摘要
采用逆转录-聚合酶链式反应(RT-PCR)方法,从牦牛睾丸组织获得Prdm9基因的cDNA的保守区序列,克隆到pMD19-T载体中。经测序确证后,将基因克隆到原核表达载体PET32a(+),转化E.coli表达菌BL21(DE3),以IPTG诱导融合蛋白的高效表达。利用Ni柱亲和层析纯化蛋白,将得到的抗原免疫日本大白兔,获得了1∶4效价的牦牛PRDM9保守序列的多克隆抗体,可用于后续试验的研究。
The conserved region of Prdm9 cDNA was obtained from yak testis by RT-PCR and cloned into pMD19-T vector.Following sequence confirmation, the cDNA was ligated with pET-32a(+)vector and then transformed into E. coli BL21(DE3)to construct expression strains.The recombinant PRDM9 protein was efficiently expressed in the form of fusion protein through induction with IPTG.After purification by Ni-NTA, the recombinant protein was injected into Japan White rabbits, and polyclonal antibody against the conserved region of PRDM9 of yak was obtained and the antiserum was collected after the titter reach 1∶4, the antibody can be used in the future research.
The conserved region of Prdm9 cDNA was obtained from yak testis by RT-PCR and cloned into pMD19-T vector.Following sequence confirmation, the cDNA was ligated with pET-32a(+)vector and then transformed into E. coli BL21(DE3)to construct expression strains.The recombinant PRDM9 protein was efficiently expressed in the form of fusion protein through induction with IPTG.After purification by Ni-NTA, the recombinant protein was injected into Japan White rabbits, and polyclonal antibody against the conserved region of PRDM9 of yak was obtained and the antiserum was collected after the titter reach 1∶4, the antibody can be used in the future research.
引文
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[12]郭晓强.PRDM9在哺乳动物基因重组热点中的作用[J].生物化学与生物物理进展,2010,37(9):929-933.
[13]Hayashi K,Yoshida K,Matsui Y.A histone H3 methyltzantfferase controls epigenetic events required for meiotic prophase[J].Nature,2005,438(7066):374-378.
[14]谭荣荣,刘明炎,龚自明,等.茶尺蠖核型多角体病毒多克隆抗体的制备及检测应用[J].湖北农业科学,2012,5(24):299-301.
[15]Hochuli E,Dobeli H,Schacher A.New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues[J].J Chromatogr,1987,411(2):177-184.
[2]Zhang QB,Li JH,Li QF,et al.Cloning and characterization of the gene encoding the bovine boule protein[J].Mol Genet Genomics,2009,28(1):67-75.
[3]李贤,李齐发,等.牦牛和犏牛Dmc1基因序列分析及睾丸组织转录水平研究[J].中国农业科学,2010,43(15):3221-3229.
[4]张旭静.牦牛和普通牛种间杂种公牛睾丸的组织学观测与研究[J].畜牧兽医学报,2001,32(4):314-318.
[5]贾荣莉.牦牛及其与黄牛杂交代睾丸的比较研究[J].四川畜牧兽医,2001,28(10):23.
[6]贾荣莉.牦牛及牦牛与黄牛杂种1-3代牛睾丸的比较组织学研究[J].黑龙江畜牧兽医,2001,(7):10-11.
[7]Borde V,Robine N,Lin W,et al.Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites[J].EMBO J,2009,28(2):99-111.
[8]Irie S,Tsujimura A,et al.Single-nucleotide polymorphisms of the PRDM9(MEISETZ)gene in patients with nonobstructive azoospermia[J].J Androl,2009,30(4):426-431.
[9]Miyamoto T,Koh E,Sakugawa N,et al.Two single nucleotide polymorphisms in PRDM9(MEISETZ)gene may be a genetic risk factor for Japanese patients with azoospermia by meiotic arrest[J].J Assist Reprod Genet,2008,25(11-12):553-557.
[10]Thomas JH,Emerson RO,Shendure J.Extraordinary molecular evolution in the PRDM9 fertility gene[J].PLoS One,2009,4(12):8505.
[11]Baudat F,Buard J,Grey C,et al.PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice[J].Science,2010,327(5967):836-840.
[12]郭晓强.PRDM9在哺乳动物基因重组热点中的作用[J].生物化学与生物物理进展,2010,37(9):929-933.
[13]Hayashi K,Yoshida K,Matsui Y.A histone H3 methyltzantfferase controls epigenetic events required for meiotic prophase[J].Nature,2005,438(7066):374-378.
[14]谭荣荣,刘明炎,龚自明,等.茶尺蠖核型多角体病毒多克隆抗体的制备及检测应用[J].湖北农业科学,2012,5(24):299-301.
[15]Hochuli E,Dobeli H,Schacher A.New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues[J].J Chromatogr,1987,411(2):177-184.