The Soluble Expression and Bioactivity of Porcine (Sus scrofa) Recombinant Interferon α/Porcine Interleukin-2 In vitro
FENG Gui-Dan1,4, YAN Ruo-Qian2,3,*, MA Zhen-Yuan2,3, CHAI Mao2,3, ZHOU Jian-Hao1, KANG Tai-Sheng5, WANG Shu-Juan2,3, YANG Hai-Bo2,3, LIU Ying2,3, XIE Cai-Hua2,3, ZHAO Xue-Li2,3, WANG Cui2,3
1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; 2 Henan Centre for Animal Diseases Control and Prevention, Zhengzhou 450008, China; 3 Key Laboratory of Surveillance and Early Warning, Prevention and Control of Major Animal Diseases of Henan Province, Zhengzhou 450008, China; 4 Shanghai Animal Disease Prevention and Control Center, Shanghai 201103, China; 5 College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471000, China
Abstract：Porcine (Sus scrofa) interferon α/interleukin-2 (PoIFN-α-linker-PoIL-2) is mostly expressed as inclusion bodies in Escherichia coli. To obtain soluble expression of active recombinant (r) PoIFN-α-linker-PoIL-2 in the E. coli system, the chimeric gene PoIFN-α-linker-PoIL-2 was synthesised by soluble modification based on the codon preference of E. coli. The modified PoIFN-α-linker-PoIL-2 chimeric gene was cloned into the expression vector pET-32a(+) for prokaryotic expression, and the expressed soluble recombinant fusion protein (rPoIFN-α-linker-PoIL-2) was purified using a nickel-chromium affinity chromatography column. The proliferative activity of rPoIFN-α-linker-PoIL-2 protein on peripheral blood T lymphocytes in vitro was detected by lymphocyte proliferation assay, could also be detected by ELISA assay using anti-PoIL-2 monoclonal antibody or anti-PoIFN-α monoclonal antibody. The antiviral bioactivity of rPoIFN-α-linker-PoIL-2 protein was tested by inhibiting the 50% appearance of cytopathic effect (CPE) of different viruses on different cell lines. The results showed that the chimeric gene PoIFN-α-linker-PoIL-2 could be efficiently expressed in E. coli. The expressed rPoIFN-α-linker-PoIL-2 protein had a molecular weight of about 55 kD. The purity of rPoIFN-α-linker-PoIL-2 protein was over 90% after purification by Ni-Cr affinity chromatography, which had significant proliferative activity on peripheral blood T lymphocytes (PBLC). Specific immune response can be detected by anti-PoIL-2 and anti-PoIFN-α monoclonal antibodies. The rPoIFN-α-linker-PoIL-2 protein had different inhibitory activities on the proliferation of different viruses in different cells. The activity units of inhibiting the proliferation of Vesicular stomatitis virus (VSV) in porcine kidney cells (PK-15) and human amniotic cells WISH were were 1.8×106 and 2.5×106 IU/mg. The active units of inhibiting the proliferation of Pseudorabies virus (PRV) and Seneca virus (SVA) on PK-15 cells were 2.2×104 and 1.3×105 IU/mg, respectively. The activity unit for inhibiting the proliferation of Porcine epidemic diarrhea virus (PEDV) in Verda Reno (Vero) was 3.2×104 IU/mg. The soluble rPoIFN-α-linker-PoIL-2 protein was obtained in this study, which could inhibit the proliferation of VSV, PRV, SVA and PEDV in different cells. This study provides basic data for further study on the activity of rPoIFN-α-linker-PoIL-2 protein in pigs.
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