犬β防御素CBD122原核表达及其抗病毒活性研究  

Prokaryotic Expression and Antiviral Activity of Canine Beta Defensin CBD122

作　　者：任艳蓉 李传峰[2] 孟春春[2] 朱杰[2] 赵建军[1,3] 刘光清 REN Yanrong;LI Chuanfeng;MENG Chunchun;ZHU Jie;ZHAO Jianjun;LIU Guangqing(Institute of Special Animals and Plant Sciences,CAAS,Changchun 130112,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]中国农业科学院特产研究所,长春130112 [2]中国农业科学院上海兽医研究所,上海200241 [3]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《中国动物传染病学报》2024年第5期135-143,共9页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(32000109,31672572);中国农业科学院基本业务费项目(2022JB01)。

摘　　要：防御素为抗菌肽家族成员,且具有良好的抗菌抗病毒功能。由于抗生素的大量使用其作用已大大降低,而防御素具有良好的抗病毒应用前景,因此本研究将犬β防御素CBD122利用大肠杆菌表达重组蛋白rCBD122,经纯化后在体外细胞上进行抗重组水泡性口炎病毒(recombinant Vesicular stomatitis virus expressing green fluorescent protein,rVSV-GFP)和犬瘟热病毒(Canine distemper virus,CDV)活性测定,从而评价重组蛋白抗病毒效果。研究结果显示,37℃诱导5 h,IPTG浓度为0.5 mmol/L时rCBD122表达量较高,且大量表达于菌体破碎后菌液上清液,纯化后表达量为0.22 mg/mL。rCBD122抗rVSV-GFP和CDV感染活性测定结果显示,rCBD122抗VSV-GFP病毒效价为1.17×10^(4)IU/mL;rCBD122抗CDV感染效价为2.34×10^(4)IU/mL。rCBD122可在24 h特异性抑制CDV野毒株SD(14)7在易感细胞上的复制水平(P<0.01),与病毒感染组相比,病毒RNA载量下降16.31倍。因此,本研究应用大肠杆菌表达系统成功表达了重组蛋白rCBD122,且其具有较好的抗病毒活性,为后续防御素作用机制研究以及新型抗病毒治疗药物研发奠定了基础。Defensins are members of antimicrobial peptide family,which have antibacterial and antiviral functions.Due to the large-scale use of antibiotics,their eff ect has been greatly reduced and defensins have good antiviral application prospects.In this study,the canine beta defensin CBD122 was expressed using Escherichia coli as the host bacteria.After purifi cation,the recombinant protein(rCBD122)was tested for its anti-recombinant vesicular stomatitis virus expressing green fl uorescent protein(rVSV-GFP)and Canine distemper virus(CDV)activity on cells in vitro.The results showed that the recombinant protein were highly expressed and released into supernatant with induction of IPTG of 0.5 mmol/L at 37℃for 5 h.The purifi ed protein concentration reached to 0.22 mg/mL.The results showed that the antiral titers of rCBD122 were 1.17×10^(4)IU/mL for VSV-GFP virus and 2.34×10^(4)IU/mL for CDV.The rCBD122 specifi cally inhibited the replication level of CDV SD(14)7 on susceptible cells at 24 h(P<0.01).Compared with the PBS control group,the viral RNA load decreased by 16.31 times.In conclusion,rCBD122 was successfully expressed in E.coli expression system in this study and had good antiviral activity.These results laid a foundation for the future study of the antiviral mechanism of defensins and development of new therapeutic drugs.

关 键 词：犬防御素 原核表达 大肠杆菌 抗病毒 活性 

分 类 号：S852.4[农业科学—基础兽医学]