| 太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备 |
| |
| 引用本文： | 吕绘倩,蒋洁兰,姜志强,毛明光.太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备[J].大连海洋大学学报,2017,32(2). |
| |
| 作者姓名： | 吕绘倩 蒋洁兰 姜志强 毛明光 |
| |
| 作者单位： | 大连海洋大学 农业部北方海水增养殖重点实验室, 辽宁省北方鱼类应用生物学与增养殖重点实室, 辽宁 大连 116023 |
| |
| 基金项目： | 国家自然科学基金资助项目，辽宁省教育厅科研项目，农业部北方海水增养殖重点实验室开放课题 |
| |
| 摘 要： | 为进一步研究太平洋鳕Gadus macrocephalus抗冻蛋白AFP4的功能,通过RT-PCR方法克隆得到太平洋鳕4型抗冻蛋白基因(AFP4)的开放阅读框(open reading frame,ORF),构建原核表达载体并优化表达条件,利用纯化的重组蛋白制备多克隆抗体,采用间接ELISA法检测抗体的效价,用Western-blot法分析重组蛋白的免疫原性。结果表明:AFP4基因编码区长度为375 bp,编码125个氨基酸;将AFP4基因的ORF与载体p ET-32a连接,构建重组体p ET-32a-AFP4,并将其转入大肠杆菌BL21(DE3)感受态细胞中;经诱导、表达和条件优化,发现该重组体在37℃、0.01 mmol/L IPTG条件下诱导3 h获得最大的表达量;对该重组蛋白进行纯化,利用纯化的蛋白免疫新西兰大白兔制备多克隆抗体,采用间接ELISA法检测该抗体效价为1∶1 600 000;Western blot分析显示,重组蛋白可以与兔抗AFP4多克隆抗体发生特异性结合,表明该重组蛋白具有免疫原性。本研究结果可为深入研究太平洋鳕AFP4蛋白功能提供理论依据。
|
| 关 键 词： | 太平洋鳕 抗冻基因 原核表达 抗体 |
Prokaryotic expression and polyclonal antibody preparation of antifreeze protein gene AFP4 in Pacific cod Gadus macrocephalus |
| |
| Authors: | L&#; Hui-qian JIANG Jie-lan JIANG Zhi-qiang MAO Ming-guang |
| |
| Institution: | Lü Hui-qian,JIANG Jie-lan,JIANG Zhi-qiang,MAO Ming-guang |
| |
| Abstract: | The open reading frame (ORF) of type Ⅳ antifreeze protein was cloned from Pacific cod Gadus macro-cephalus using RT-PCR to investigate the function of type Ⅳ antifreeze protein. The result showed that the ORF of AFP4 had 375 bp in length, encoding a polypeptide of 125 amino acids. The recombinant pET-32a-AFP4 was constructed by linking the AFP4 ORF with the pET - 32a vector, and transformed into Escherichia coli BL21 (DE3). The expressing conditions of the recombinant plasmid were optimized under various concentrations of ther-mal hysteresis protein (THP), induction time and temperature with the optimal expressing induction conditions of 0. 01 mmol/ L IPTG, 3 h and 37 ℃. The recombinant protein was purified and used to immunize New Zealand white rabbit. The polyclonal antibody was obtained and tested using indirect ELISA, with the titer of 1 :1600000. The specific binding between recombinant protein and Rabbit anti-AFP4 polyclonal antibody indicated that the recombinant protein had high immunogenicity. The findings will provide the foundations for further under-standing of AFP4 functions in Pacific cod. |
| |
| Keywords: | Gadus macrocephalus antifreeze gene prokaryotic expression antibody |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
