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当前位置: 首页 > 文章 > 青鱼Dazl基因的原核表达及其多克隆抗体制备 淡水渔业 2019,49 (3) 27-31
Position: Home > Articles > Prokaryotic expression and polyclonal antibody production of Dazl gene in Mylopharyngodon piceus Freshwater Fisheries 2019,49 (3) 27-31

青鱼Dazl基因的原核表达及其多克隆抗体制备

作  者：
王艺舟;潘启华;王乾;夏必琳;罗君志;方健;邓羽;廖明聪;陈天圣
单  位：
华中农业大学水产学院
关键词：
青鱼(Mylopharyngodon piceus);Dazl;原核表达;多克隆抗体
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.
译  名：
Prokaryotic expression and polyclonal antibody production of Dazl gene in Mylopharyngodon piceus
作  者：
WANG Yi-zhou;PAN Qi-hua;WANG Qian;XIA Bi-lin;LUO Jun-zhi;FANG Jian;DENG Yu;LIAO Ming-cong;CHEN Tian-sheng;Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture;College of Fisheries,Huazhong Agricultural University;Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province;
关键词：
Mylopharyngodon piceus;;Dazl;;prokaryotic expression;;polyclonal antibody
摘  要：
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.

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