青鱼Dazl基因的原核表达及其多克隆抗体制备
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摘要
实验进行了青鱼(Mylopharyngodon piceus)Dazl基因的原核表达,兔抗青鱼源Dazl多克隆抗体的制备及抗体的特异性验证。首先将青鱼Dazl基因的编码区利用重组表达引物从pCS2-MpDazl质粒中扩增出来,经酶切后连接到pET-28a载体中,构建pET-28a-MpDazl重组表达载体。然后将pET-28a-MpDazl重组质粒转化至大肠杆菌(Escherichia coli)BL21中,利用异丙基-β-d-硫代半乳糖苷(IPTG)诱导表达,获得分子量为27 kD的Dazl重组蛋白。将纯化的Dazl重组蛋白作为抗原免疫家兔制备多克隆抗体,抗体的效价和特异性通过ELISA法和Western blot检测。结果:成功构建重组表达载体pET-28a-MpDazl;以0.5 mmol/L IPTG在37℃条件下诱导4 h可获得高效表达的Dazl重组蛋白;制备的兔抗青鱼Dazl多克隆抗体能够特异性识别原核表达Dazl蛋白、青鱼卵巢的内源Dazl蛋白以及细胞中过表达的Dazl蛋白,并证实了青鱼Dazl蛋白在性腺中表达的特异性。
The prokaryotic expression of the recombinant Dazl protein and generation of the rabbit anti-Dazl polyclonal antibody in Mylopharyngodon piceu were investigated in this study. The amplified MpDazl coding sequences were inserted into pET-28 a to generate the pET-28 a-MpDazl. The recombinant vector was transformed into Escherichia coli BL21(DE3) pLysS. Meanwhile, the Dazl protein was induced by IPTG. After large preparation, the MpDazl protein was purified and applied to produce the antibody. The specificity of the antibody was examined by ELISA and Western blot. The results indicated that the recombinant Dazl protein could be highly expressed under the condition of 37 ℃ with 0.5 mmol/L IPTG induction for 4 h. Moreover, the polyclonal antibody specifically recognized the induced Dazl protein in E. coli, the endogenous MpDazl protein in the ovary of black carp, and the ectopic expressed MpDazl proteins in fish cells. As a conclusion, the Dazl antibody validates the gonadal expression of Dazl protein and provides a excellent tool to investigate the role of Dazl in black carp germ cells.
The prokaryotic expression of the recombinant Dazl protein and generation of the rabbit anti-Dazl polyclonal antibody in Mylopharyngodon piceu were investigated in this study. The amplified MpDazl coding sequences were inserted into pET-28 a to generate the pET-28 a-MpDazl. The recombinant vector was transformed into Escherichia coli BL21(DE3) pLysS. Meanwhile, the Dazl protein was induced by IPTG. After large preparation, the MpDazl protein was purified and applied to produce the antibody. The specificity of the antibody was examined by ELISA and Western blot. The results indicated that the recombinant Dazl protein could be highly expressed under the condition of 37 ℃ with 0.5 mmol/L IPTG induction for 4 h. Moreover, the polyclonal antibody specifically recognized the induced Dazl protein in E. coli, the endogenous MpDazl protein in the ovary of black carp, and the ectopic expressed MpDazl proteins in fish cells. As a conclusion, the Dazl antibody validates the gonadal expression of Dazl protein and provides a excellent tool to investigate the role of Dazl in black carp germ cells.
引文
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[18]段小明,唐头生,段建元.青鱼人工繁殖技术[J].科学养鱼,2010,(10):10-11.
[19]马徐发.三种以青鱼为主要养殖对象的池塘养殖模式[J].养殖与饲料,2011,(09):19-20.
[20] Xue T,Wang Y Z,Pan Q H,et al.Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus [J].J Fish Dis,2018,41(2):365-374.
[21]张雪萍,曾令兵,陈倩,等.青鱼鳍条组织细胞系的建立及其生物学特性[J].淡水渔业,2016,46(2):3-9.
[22]张建铭,吴志强,胡茂林.赣江峡江段四大家鱼资源现状的研究[J].水生态学杂志,2010,31(1):34-37.
[23]李玲玉,方健,于淼,等.青鳉prdm14的原核表达,多克隆抗体制备及其应用[J].水生生物学报,2017,41(4):748-754.
[24]于淼,方健,李玲玉,等.团头鲂转录因子Nanog的原核表达及其多克隆抗体制备[J].水产学报,2017,(11):1-11.
[25]刘平,张昀,郑喜邦,等.山羊SOX2多克隆抗体制备[J].中国农业科学,2012,45(1):178-183.
[26] Bhat N,Hong Y.Cloning and expression of boule and Dazl in the Nile tilapia(Oreochromis niloticus) [J].Gene,2014,540(2):140-145.
[2] Carani C,Gromoll J,Brinkworth M H,et al.cynDAZLA:a cynomolgus monkey homologue of the human autosomal DAZ gene [J].Mol Hum Reprod,1997,3(6):479-483.
[3] Eberhart C G,Maines J Z,Wasserman S A.Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia [J].Nature,1996,381(6585):783-785.
[4] Saxena R,Brown L G,Hawkins T,et al.The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed,repeatedly amplified and pruned [J].Nat Genet,1996,14(3):292-299.
[5] Yen P H,Chai N N,Salido E C.The human DAZ genes,a putative male infertility factor on the Y chromosome,are highly polymorphic in the DAZ repeat regions [J].Mamm Genome,1997,8(10):756-759.
[6] Xu E Y,Moore F L,Pera R A R.A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans [J].Proc Natl Acad Sci,2001,98(13):7414-7419.
[7] Tung J Y,Rosen M P,Nelson L M,et al.Variants in Deleted in AZoospermia-Like(DAZL) are correlated with reproductive parameters in men and women [J].Hum Genet,2006,118(6):730-740.
[8] Houston D W,King M L.A critical role for XDazl,a germ plasm-localized RNA,in the differentiation of primordial germ cells in Xenopus [J].Development,2000,127(3):447-456.
[9] Vogel T,Speed R M,Ross A,et al.Partial rescue of the Dazl knockout mouse by the human DAZL gene [J].Mol Hum Reprod,2002,8(9):797-804.
[10] Maegawa S,Yasuda K,Inoue K.Maternal mRNA localization of zebrafish DAZ-like gene [J].Mech Dev,1999,81(1):223-226.
[11] Xu H,Li M,Gui J,et al.Cloning and expression of medaka Dazl during embryogenesis and gametogenesis [J].Gene Expr Patt,2007,7(3):332-338.
[12]姜佳君.牙鲆(Paralichthys olovaceus) Dazl基因的克隆、表达和功能的初步研究[D].青岛:中国海洋大学,2015.
[13] Peng J X,Xie J L,Zhou L,et al.Evolutionary conservation of Dazl genomic organization and its continuous and dynamic distribution throughout germline development in gynogenetic gibel carp [J].J Exp Zool Part B:Mol Dev Evol,2009,312(8):855-871.
[14]张红.半滑舌鳎性别相关基因DAZL和WT1A的克隆与表达分析[D].青岛:中国海洋大学,2013.
[15] Dwarakanath M,Lim M,Xu H,et al.Differential expression of boule and Dazl in adult germ cells of the Asian seabass [J].Gene,2014,549(2):237-242.
[16] Ye H,Li C J,Yue H M,et al.Differential expression of fertility genes boule and Dazl in Chinese sturgeon(Acipenser sinensis),a basal fish [J].Cell Tissue Res,2015,360(2):413-425.
[17]郭云峰,赵文武.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017.
[18]段小明,唐头生,段建元.青鱼人工繁殖技术[J].科学养鱼,2010,(10):10-11.
[19]马徐发.三种以青鱼为主要养殖对象的池塘养殖模式[J].养殖与饲料,2011,(09):19-20.
[20] Xue T,Wang Y Z,Pan Q H,et al.Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus [J].J Fish Dis,2018,41(2):365-374.
[21]张雪萍,曾令兵,陈倩,等.青鱼鳍条组织细胞系的建立及其生物学特性[J].淡水渔业,2016,46(2):3-9.
[22]张建铭,吴志强,胡茂林.赣江峡江段四大家鱼资源现状的研究[J].水生态学杂志,2010,31(1):34-37.
[23]李玲玉,方健,于淼,等.青鳉prdm14的原核表达,多克隆抗体制备及其应用[J].水生生物学报,2017,41(4):748-754.
[24]于淼,方健,李玲玉,等.团头鲂转录因子Nanog的原核表达及其多克隆抗体制备[J].水产学报,2017,(11):1-11.
[25]刘平,张昀,郑喜邦,等.山羊SOX2多克隆抗体制备[J].中国农业科学,2012,45(1):178-183.
[26] Bhat N,Hong Y.Cloning and expression of boule and Dazl in the Nile tilapia(Oreochromis niloticus) [J].Gene,2014,540(2):140-145.