| 青鱼Dazl基因的原核表达及其多克隆抗体制备 |
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| 引用本文： | 王艺舟,潘启华,王乾,夏必琳,罗君志,方健,邓羽,廖明聪,陈天圣.青鱼Dazl基因的原核表达及其多克隆抗体制备[J].淡水渔业,2019,49(3):27-31. |
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| 作者姓名： | 王艺舟 潘启华 王乾 夏必琳 罗君志 方健 邓羽 廖明聪 陈天圣 |
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| 作者单位： | 华中农业大学水产学院,农业部淡水生物繁育重点实验室,武汉430070;华中农业大学水产学院,农业部淡水生物繁育重点实验室,武汉430070;水产高效健康生产湖南省协同创新中心,湖南常德415000 |
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| 基金项目： | 国家自然科学基金;国家自然科学基金;创新基金 |
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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。
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| 关 键 词： | 青鱼( Mylopharyngodon piceus ) DAZL 原核表达 多克隆抗体 |
Prokaryotic expression and polyclonal antibody production of Dazl gene in Mylopharyngodon piceus |
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| WANG Yi-zhou,PAN Qi-hua,WANG Qian,XIA Bi-lin,LUO Jun-zhi,FANG Jian,DENG Yu,LIAO Ming-cong,CHEN Tian-sheng.Prokaryotic expression and polyclonal antibody production of Dazl gene in Mylopharyngodon piceus[J].Freshwater Fisheries,2019,49(3):27-31. |
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| Authors: | WANG Yi-zhou PAN Qi-hua WANG Qian XIA Bi-lin LUO Jun-zhi FANG Jian DENG Yu LIAO Ming-cong CHEN Tian-sheng |
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| Institution: | (College of Fisheries,Huazhong Agricultural University, Wuhan 430070, China;Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Changde 41500,Hunan,China) |
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| Abstract: | 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-28a to generate the pET-28a- 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. |
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| Keywords: | Mylopharyngodon piceus Dazl prokaryotic expression polyclonal antibody |
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