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2025年8月14日 星期四
农业生物技术学报  2022, Vol. 30 Issue (4): 809-816    DOI: 10.3969/j.issn.1674-7968.2022.04.019
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
鸡HNF 3β重组蛋白在大肠杆菌中的高效表达及其抗体制备
张景, 潘玲, 毛威威, 周夏燕, 郁建锋*, 徐璐, 顾志良*
常熟理工学院, 常熟 215500
High-level Expression of Chicken (Gallus gallus) HNF 3β Recombinant Protein in Escherichia coli and Preparation of Its Antibody
ZHANG Jing, PAN Ling, MAO Wei-Wei, ZHOU Xia-Yan, YU Jian-Feng*, XU Lu, GU Zhi-Liang*
Changshu Institute of Technology, Changshu 215500, China
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摘要 肝核因子3β (hepatocyte nuclear factor 3β, HNF 3β)作为一个重要的转录调控因子,参与哺乳动物的前肠胚和肝脏发育以及成体肝内的能量代谢,而HNF 3β在鸡(Gallus gallus)中的功能还不清楚。为研究鸡HNF 3β在肝内糖脂代谢中的作用提供有效的抗体,本研究优化合成了鸡Hnf 3β基因编码区片段,以pET 30a表达载体在大肠杆菌(Escherichia coli)中进行重组蛋白HNF 3β的表达,用6 mol/L尿素变性包涵体,Ni2+对重组蛋白进行亲和层析,并以快速稀释法复性重组蛋白,以离心超滤法收集、浓缩目的蛋白;取纯化的HNF 3β重组蛋白和弗氏佐剂乳化后免疫兔(Oryctolagus cuniculus),制备鸡HNF 3β的抗血清,以酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)测定抗血清效价后,用Protein A法纯化抗体;通过检测鸡肝癌细胞系(leghorn male hepatoma, LMH)中HNF 3β蛋白的表达情况验证所制备抗体的效果。结果表明,优化合成的Hnf 3β基因片段在大肠杆菌中实现了重组蛋白的高效表达;在6 mol/L尿素的变性条件下,Ni2+亲和层析一步法获得纯度大于95%的重组蛋白,结合快速稀释法获得了可溶性的重组蛋白HNF 3β;经重组蛋白4次免疫后,抗血清效价超过1∶64 000;免疫印迹结果显示,制备的抗体能特异性的检测出LMH细胞中HNF 3β蛋白的表达。综上所述,本研究利用制备的高纯度重组蛋白HNF 3β免疫兔获得了高特异性和高效价的多克隆抗体,可以为检测鸡HNF 3β蛋白分子及研究其功能提供可靠的抗体。
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张景
潘玲
毛威威
周夏燕
郁建锋
徐璐
顾志良
关键词 肝核因子3β(HNF 3β)重组蛋白免疫抗体    
Abstract:Hepatocyte nuclear factor 3β (HNF 3β) is an important transcription factor involved in the foregut and liver development in mammalian and energy metabolism in adult liver. However, the function of HNF 3β in chicken (Gallus gallus) is still unknown. In order to provide effective antibodies for studying the roles of HNF 3β in glucose and lipid metabolism in chicken liver, the encoding region segment of chicken Hnf 3β was optimized according to the codon preference in Escherichia coli and synthesized. Then this segment was cloned into pET 30a and the HNF 3β recombinant protein was expressed in E. coli. The recombinant protein was purified from the denatured inclusion body with 6 mol/L urea using Ni2+ affinity chromatography. The denatured protein was diluted into the refolding buffer at ration of 1∶20 for protein renaturation. The purified recombinant protein emulsified with Freund's adjuvant was used to immunize rabbits (Oryctolagus cuniculus) to prepare chicken HNF 3β antiserum. The antiserum titers were determined using the enzyme linked immunosorbent assay (ELISA) method, and the antibody was purified by the Protein A method. The effects of the antibody were verified by the detection of HNF 3β protein expressed in the hepatocellular carcinoma cell line, leghorn male hepatoma (LMH). The results showed that the recombinant protein HNF 3β was effectively express in E. coli. after the sequence of the chicken Hnf 3β was optimized. The recombinant protein HNF 3β was expressed as the inclusion bodies in E. coli. The recombinant protein was purified by Ni2+ affinity chromatography from the inclusion bodies denatured with 6 mol/L urea. The soluble recombinant protein HNF 3β was obtained with a purity of more than 95% after a denaturing and renaturing procedure. The titer of the antiserum from rabbits was over 1∶64 000 after immunization with the recombinant protein for 4 times by ELISA detection. The polyclonal antibodies of HNF 3β were then purified with the protein A affinity chromatography. The western blot results showed that the prepared antibody could specifically detect the expression of HNF 3β protein in LMH cells. The expression of HNF 3β was more than 5 times in LMH cells transfected with the pcDNA3.1(+)-ggaHnf 3β for 48 h in comparison with the control. In conclusion, the high specificity and affinity polyclonal antibodies against HNF 3β were obtained from the rabbits immunized with the purified recombinant protein HNF 3β, which contribute to detect the chicken HNF 3β and provide a tool to investigate its roles in chicken metabolism regulation.
Key wordsChicken    Hepatocyte nuclear factor 3β(HNF 3β)    Recombinant protein    Immunization    Antibody
收稿日期: 2021-08-11     
ZTFLH:  S831.2  
基金资助:国家自然科学基金(31772593); 江苏省自然科学基金(BK20191476)
通讯作者: *csyjf@cslg.edu.cn;zhilianggu88@hotmail.com   
引用本文:   
张景, 潘玲, 毛威威, 周夏燕, 郁建锋, 徐璐, 顾志良. 鸡HNF 3β重组蛋白在大肠杆菌中的高效表达及其抗体制备[J]. 农业生物技术学报, 2022, 30(4): 809-816.
ZHANG Jing, PAN Ling, MAO Wei-Wei, ZHOU Xia-Yan, YU Jian-Feng, XU Lu, GU Zhi-Liang. High-level Expression of Chicken (Gallus gallus) HNF 3β Recombinant Protein in Escherichia coli and Preparation of Its Antibody. 农业生物技术学报, 2022, 30(4): 809-816.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.04.019     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I4/809
 
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