重组鸡白介素2和干扰素α融合蛋白的表达及生物学活性的检测  

Expression and biological activity detection of recombinant chicken interleukin-2 and interferon-αfusion protein

作　　者：苗天姿 赵款 雷白时[1,2] 张武超 庞敬红 梁飞 袁万哲 汪恩强[1] MIAO Tianzi;ZHAO Kuan;LEI Baishi;ZHANG Wuchao;PANG Jinghong;LIANG Fei;YUAN Wanzhe;WANG Enqiang(College of Animal Medicine,Hebei Agriculture University,Baoding 071001,China;Hebei Veterinary Biotechnology Engineering Technology Innovation Center,Baoding 071001,China;Hebei Borui Animal Pharmaceutical Co.,Ltd,Zhengding 050800,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术工程技术创新中心,河北保定071001 [3]河北伯瑞动物药业有限公司,河北正定050800

出　　处：《河北农业大学学报》2021年第6期85-91,共7页Journal of Hebei Agricultural University

基　　金：2020年度石家庄市重点研发计划项目(201500182A).

摘　　要：为表达鸡的白介素-2(Interleukin-2,IL-2)和干扰素α(Interferon-α,IFN-α)融合蛋白,并进一步探究融合蛋白的生物学活性,本试验根据GenBank登录的鸡IL-2和IFN-α基因序列,使用linker连接成为嵌合基因,合成pET-32a-rChIL-2-IFN-α原核表达载体,转化至大肠杆菌BL21(DE3)经IPTG诱导表达。通过MTT法检测重组融合蛋白促鸡外周血淋巴细胞(PBLC)增殖活性;通过细胞病变抑制法检测重组融合蛋白在鸡胚成纤维细胞(CEF)上的抗病毒活性;通过RT-qPCR检测了融合蛋白对干扰素刺激基因和内源性干扰素的作用。结果表明,重组融合蛋白促PBLC增殖活性效果显著,在CEF上具有抗VSV活性;重组融合蛋白能够显著地上调CEF中5种干扰素刺激基因和内源性干扰素的mRNA转录水平。综上所述,本试验表达的重组融合蛋白具有促淋巴细胞增殖活性和抗病毒作用,试验结果为进一步探究融合蛋白在鸡体内的活性奠定了基础。In order to express the fusion proteins interleukin-2(IL-2)and Interferon-α(IFN-α),and to further explore the biological activity of the fusion protein,this study was conducted according to the gene sequences of chicken IL-2 and IFN-αlogged in GenBank.The prokaryotic expression vector pET-32a-rChIL-2-IFN-αwas synthesized by linker ligating into chimeric gene and transformed into E.coli BL21(DE3)on IPTG induced expression.MTT assay was used to detect the proliferation activity of the recombinant fusion protein in chicken peripheral blood lymphocytes(PBLC). The antiviral activity of recombinant fusion protein on chicken embryo fibroblast (CEF) was detected by cytopathic inhibition. The effects of recombinant fusion protein on interferon stimulating gene and endogenous interferon were detected by RT-qPCR. The results showed that the recombinant fusion protein promoted the proliferation of PBLC significantly, and had anti-VSV activity on CEF. The recombinant fusion protein significantly upregulated the mRNA transcription levels of 5 interferon-stimulating genes and endogenous interferon in CEF. In conclusion, the recombinant fusion protein expressed in this study has the activity of promoting lymphocyte proliferation and antiviral effect, and the test results lay a foundation for further exploring the activity of fusion protein in chickens.

关 键 词：鸡IL-2 鸡IFN-α 融合蛋白 抗病毒 干扰素刺激基因 

分 类 号：S852[农业科学—基础兽医学] Q789[农业科学—兽医学]