农业生物技术学报2024，Vol.32Issue(3)：595-604,10.DOI:10.3969/j.issn.1674-7968.2024.03.010
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
The Soluble Expression and Bioactivity of Porcine(Sus scrofa)Recombinant Interferon α/Porcine Interleukin-2 In vitro
摘要
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.关键词
猪α干扰素/猪白细胞介素-2/嵌合基因/可溶性表达/体外活性Key words
Porcine interferon α/Porcine interleukin-2/Chimeric gene/Soluble expression/In vitro activity分类
农业科技引用本文复制引用
冯桂丹,谢彩华,赵雪丽,王翠,闫若潜,马震原,柴茂,周建浩,康台生,王淑娟,杨海波,刘影..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[J].农业生物技术学报,2024,32(3):595-604,10.基金项目
河南省重点研发专项(231111111300) （231111111300）
河南省重大科技专项(221100110600) （221100110600）
河南省现代农业产业技术体系(HARS-22-12-T) （HARS-22-12-T）
河南省科技攻关项目(232102110089) （232102110089）
