重组猪α干扰素/白细胞介素-2的可溶性表达及体外活性研究

doi:10.3969/j.issn.1674-7968.2024.03.010

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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.

Key words： Porcine interferon α Porcine interleukin-2 Chimeric gene Soluble expression In vitro activity

Received: 16 November 2022

ZTFLH:	S852.4
	S859.79

Corresponding Authors: * qingfengyimeng118@126.com

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	FENG Gui-Dan
	YAN Ruo-Qian
	MA Zhen-Yuan
	CHAI Mao
	ZHOU Jian-Hao
	KANG Tai-Sheng
	WANG Shu-Juan
	YANG Hai-Bo
	LIU Ying
	XIE Cai-Hua
	ZHAO Xue-Li
	WANG Cui

Cite this article:

FENG Gui-Dan,YAN Ruo-Qian,MA Zhen-Yuan, et al. The Soluble Expression and Bioactivity of Porcine (Sus scrofa) Recombinant Interferon α/Porcine Interleukin-2 In vitro [J]. 农业生物技术学报, 2024, 32(3): 595-604.

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https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2024.03.010 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2024/V32/I3/595

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