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.
[1] 王中亮, 胡悦, 郁建锋, 等. 2019. 鸡HNF4α蛋白的原核表达、纯化及特异性抗体制备[J]. 农业生物技术学报, 27(05): 927-935.
(Wang Z L, Hu Y, Yu J F, et al.2019. Prokaryotic expression, purification and specific antibody preparation of chicken (Gallus gallus) HNF4α Protein[J]. Journal of Agricultural Biotechnology, 27(05): 927-935.)
[2] 郁建锋, 张芸, 王中亮, 等. 2018. 鸡HNF1α在大肠杆菌中的表达及其纯化[J]. 农业生物技术学报, 26(03): 469-475.
(Yu J F, Zhang Y, Wang Z L, et al.2018. Chicken (Gallus gallus) HNF1α expression in Escherichia coli and its purification[J]. Journal of Agricultural Biotechnology, 26(03): 469-475.)
[3] 张春晨, 胡双艳, 阮海华. 2020. 人源溶菌酶在大肠杆菌中的表达与复性研究[J]. 生物技术通报, 36(03): 153-161.
(Zhang C C, Hu S Y, Ruan H H.2020. Expression and renaturation of recombinant human lysozyme in Escherichia coli[J]. Biotechnology Bulletin, 36(03): 153-161.)
[4] Friedman J R, Kaestner K H.2006. The Foxa family of transcription factors in development and metabolism[J]. Cellular and Molecular Life Sciences, 63(19-20): 2317-2328.
[5] Hashemzadeh M S, Mohammadi M, Ghaleh H E G, et al.2021. Expression, solubilization, refolding and final purification of recombinant proteins as expressed in the form of "Classical Inclusion Bodies" in E. coli[J]. Protein & Peptide Letters, 28(2): 122-130.
[6] Hayat S M G, Farahani N, Golichenari B, et al.2018. Recombinant protein expression in Escherichia coli (E. coli): what we need to know[J]. Current Pharmaceutical Design, 24(6): 718-725.
[7] Hegardt F G.1999. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: A control enzyme in ketogenesis[J]. Biochemical Journal, 338(Pt 3): 569-582.
[8] Kanaki M, Kardassis D.2017. Regulation of the human lipoprotein lipase gene by the forkhead box transcription factor FOXA2/HNF-3beta in hepatic cells[J]. Biochimica et Biophysica Acta, 1860(3): 327-336.
[9] Kanaki M, Tiniakou I, Thymiakou E, et al.2017. Physical and functional interactions between nuclear receptor LXRα and the forkhead box transcription factor FOXA2 regulate the response of the human lipoprotein lipase gene to oxysterols in hepatic cells[J]. Biochimica et Biophysica Acta-Gene Regulatory Mechanisms, 1860(8): 848-860.
[10] Kelleher A M, Peng W, Pru J K, et al.2017. Forkhead box a2 (FOXA2) is essential for uterine function and fertility[J]. Proceedings of the National Academy of Sciences of the USA, 114(6): E1018-E1026.
[11] Klermund L, Riederer A, Groher A, et al.2015. High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli[J]. Protein Expression and Purification, 111: 36-41.
[12] Lee C S, Friedman J R, Fulmer J T, et al.2005. The initiation of liver development is dependent on Foxa transcription factors[J]. Nature, 435(7044): 944-947.
[13] Li Y Y, Wang X G, Yu J F, et al.2016. MiR-122 targets the vanin 1 gene to regulate its expression in chickens[J]. Poultry Science, 95(5): 1145-1150.
[14] Louet J F, May C L, Pégorier J P, et al.2001. Regulation of liver carnitine palmitoyltransferase I gene expression by hormones and fatty acids[J]. Biochemical Society Transactions, 29(Pt 2): 310-316.
[15] Mehri N, Jamshidizad A, Ghanei Z, et al.2021. Optimizing the expression and solubilization of an E. coli-produced leukemia inhibitory factor for anti-LIF antibody production and use thereof for contraception in mice[J]. Molecular Biotechnology, 63(12):1169-1182.
[16] Schrem H, Klempnauer J, Borlak J.2002. Liver-enriched transcription factors in liver function and development. Part I: The hepatocyte nuclear factor network and liver-specific gene expression[J]. Pharmacological Reviews, 54(1): 129-158.
[17] Singh S M, Panda A K.2005. Solubilization and refolding of bacterial inclusion body proteins[J]. Journal of Bioscience and Bioengineering, 99(4): 303-310.
[18] Wolfrum C, Asilmaz E, Luca E, et al.2004. Foxa2 regulates lipid metabolism and ketogenesis in the liver during fasting and in diabetes[J]. Nature, 432(7020): 1027-1032.
