猫白介素18基因的克隆、表达及其产物的生物学活性分析  

Gene cloning,soluble expression and bioactivity analysis of feline interleukin-18

作　　者：李华 汉武娇 牛超 潘荣辉 姜艳平[1] 崔文[1] 李一经[1,2,3] 徐义刚 LI Hua;HAN Wu-jiao;NIU Chao;PAN Rong-hui;JIANG Yan-ping;CUI Wen;LI Yi-jing;XU Yi-gang(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development,Harbin 150030,China;Animal Disease Control and Prevention Centre of Jilin Province,Changchun130062,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]黑龙江省动物疾病防控技术与制剂创制重点实验室,黑龙江哈尔滨150030 [3]吉林省动物疫病预防控制中心,吉林长春130062

出　　处：《中国预防兽医学报》2020年第6期612-616,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0501003)。

摘　　要：白介素18(IL-18)可以诱导IFN-γ分泌,促进T、B淋巴细胞的增殖分化,具有良好的抗病毒活性。为探究猫IL-18的生物学活性,本研究采用水泡性口炎病毒(VSV)联合poly I:C共同刺激实验猫,采用常规方法分离猫脾淋巴细胞,提取总RNA,经RT-PCR方法获得大小为579 bp的猫IL-18基因。构建了可溶性表达猫IL-18的重组大肠杆菌pCold-IL-18/BL21,经IPTG诱导,SDS-PAGE分析结果显示,重组菌表达了约70 ku的重组蛋白。重组蛋白经3C蛋白酶处理后并通过Ni柱亲和纯化,获得了可溶性表达的重组猫IL-18(rIL-18)。取BALB/c小鼠和猫的脾淋巴细胞,经不同浓度rIL-18刺激,采用MTT法检测rIL-18促进淋巴细胞增殖活性,结果显示可溶性表达的rIL-18能够有效地促进淋巴细胞增殖;采用细胞病变抑制法检测rIL-18协同干扰素的抗病毒效果,结果显示rIL-18联合临床用干扰素可显著增强抗病毒效果。本研究为治疗猫病毒性疾病奠定了物质基础。Interleukin 18(IL-18)can induce IFN-γsecretion,promote the proliferation and differentiation of T and B lymphocytes,and has good antiviral activity.In this study,in order to explore the biological activity of feline IL-18,a combination of vesicular stomatitis virus(VSV)and poly I:C was used to stimulate the cat,followed by isolation of spleen lymphocyte and total RNA extraction.The feline IL-18 gene was amplified by RT-PCR assay with the size of 579 bp.Subsequently,a recombinant E.coli pCold-IL-18/BL21 was constructed and the feline IL-18 was expressed in a soluble form.The recombinant E.coli was induced by IPTG and the SDS-PAGE analysis showed that the fusion protein was expressed with the size of 70 ku.Then,the fusion protein was treated with 3C protease and purified by Ni column affinity,the recombinant cat IL-18(rIL-18)was obtained.The different concentrations of rIL-18 were used to stimulated on the spleen lymphocytes of BALB/c mice and cats,the proliferation promoting activity of cat rIL-18 was detected by MTT method.The results showed that soluble expression of feline rIL-18 could effectively promoted the proliferation of lymphocytes.Lastly,the antiviral effect of cat rIL-18 combined with IFN was detected by cytopathic inhibition method.The results showed that the antiviral effect could be significantly enhanced by the cat rIL-18 combined with the clinical interferon.This study provided a material basis for the treatment of feline viral diseases.

关 键 词：猫白介素18 可溶性表达 生物活性 

分 类 号：S852.4[农业科学—基础兽医学]