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| 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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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.
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Received: 11 August 2021
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Corresponding Authors:
*csyjf@cslg.edu.cn; zhilianggu88@hotmail.com
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