Prokaryotic Expression, Purification and Specific Antibody Preparation of Chicken (Gallus gallus) HNF4α Protein
WANG Zhong-Liang1, 2, HU Yue2, YU Jian-Feng2, HEN Jue2, MA Li-Chen2, CHEN Chi-Chi2, YAO Wen1, *, GU Zhi-Liang2, *
1 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2 School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China
Abstract：Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily and extensively controls the basal expression of many genes involved in glucose, cholesterol and fatty acid metabolism pathways.In order to obtain chicken (Gallus gallus) HNF4α specific antibody, this study firstly optimized and synthesized chicken HNF4α gene according to the codon preference of Escherichia coli, and constructed the recombinant vector pET-30a(+)-HNF4α for prokaryotic expression of HNF4α.Next, the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) to induce expression of HNF4α protein.After optimizing the induction temperature and isopropyl-β-dthiogalactoside (IPTG) concentration, the expression product was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE.The HNF4α protein was purified by Ni-NTA agarose columand and then immunized the New Zealand rabbit (Oryctolagus cuniculus) to prepare the polyclonal antibody against chicken HNF4α.The antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA), and the expression of HNF4α in chicken tissues was further analyzed by the obtained antibody.The results showed that the codon-optimized pET-30a(+)-HNF4α was highly expressed in Escherichia coli BL21 (DE3), and the soluble protein was obtained under induction of 0.5 mmol/L IPTG for 4 h at 37 ℃ .The expressed product was purified by Ni-NTA agarose column , and a fusion protein having a size of about 51.6 kD and a purity of more than 90% was obtained by SDS-PAGE and Western blot.The polyclonal antibody titer was 1∶512 000 by ELISA.The antibody was used for detection of HNF4α in different organs of chicken, and the results showed that the HNF4α was highly expressed in the liver and kidney, and expressed in small amounts in the small intestine and testis, but hardly expressed in other tissues.In summary, this study successfully prepared chicken HNF4α antibody with high titer and specificity, which provided basic data for further investigation of chicken HNF4α gene function.
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