摘要
为制备重组山羊干扰素-τ(rGoIFN-τ)并检测其在猫肾细胞(F81)中的抗病毒活性,本研究根据大肠杆菌密码子偏好性优化合成GoIFN-τ基因后,将该基因克隆至pColdII载体,获得重组质粒pColdII-rGoIFN-τ,经鉴定正确后将其转化大肠杆菌Rosetta(DE3),经IPTG诱导表达后SDS-PAGE鉴定,利用镍离子亲和层析柱纯化蛋白,纯化的rGoIFN-τ经western blot检测后采用BCA法测定蛋白浓度。结果显示,rGoIFN-τ在大肠杆菌DE3中得到可溶性高效表达,表达量占菌体总蛋白的28.70%,纯化后蛋白浓度为100 mg/L,且具有良好的反应性。采用CCK8法检测rGoIFN-τ对F81细胞活力的影响,结果显示,其在0.0055 ng/mL~5500 ng/mL时对细胞的活力基本无影响。以水疱性口炎病毒(VSV)为模式病毒感染已与rGoIFN-τ共孵育24 h后的F81细胞,通过微量细胞病变抑制法检测rGoIFN-τ在F81/VSV系统中的抗病毒活性,结果显示,rGoIFN-τ抗VSV活性单位数为4.55×105 U/mg。经不同浓度的rGoIFN-τ(55 ng/mL、550 ng/mL、5500 ng/mL)共孵育后的F81细胞分别感染VSV,经Reed-Muench法和荧光定量PCR法检测rGoIFN-τ的抗病毒能力,结果显示,感染24 h和48 h后rGoIFN-τ可显著抑制VSV的病毒滴度和VSV-L基因转录水平(P<0.05),尤其是5500 ng/mL rGoIFN-τ的作用最佳。综上所述,本研究实现了rGoIFN-τ原核可溶性高效表达,并首次证实其在猫F81细胞中具有较好的抗病毒活性,为进一步研究rGoIFN-τ在猫抗病毒性感染中的作用奠定了基础。
In order to prepare recombinant protein of goat interferon-τ(rGoIFN-τ)and evaluate its antiviral activity on feline cells(F81 cells),the GoIFN-τgene was optimized and synthesized according to the codon preference of Escherichia coli.The gene was cloned into the pColdII vector to obtain the recombinant plasmid.After identification,the positive recombinant plasmid pColdII-rGoIFN-τwas transformed into Escherichia coli Rosetta(DE3)cells.After induction by IPTG,and the protein was determined by SDS-PAGE and purified by nickel column affinity tags.The purified rGoIFN-τprotein was detected by western blot.The concentration of obtaining protein was measured by BCA method.The results showed that the rGoIFN-τwas expressed very well in the soluble form in DE3 cells,and its amount was accounted for 28.70%of the total bacterial protein.The rGoIFN-τwith the purity of 90%was obtained and the concentration was 100mg/L,it has good reactivity.The effect of rGoIFN-τon the activity of F81 cells was detected with CCK8 method.The results showed that the rGoIFN-τwith the concentration between 0.0055ng/mL and 5500ng/mL were not significantly inhibited the activity of F81 cells with CCK8 method.The F81 cells were incubated with rGoIFN-τfor 24 hours following the infection of vesicular stomatitis virus(VSV).The antiviral activity was evaluated by microcytopathic-inhibiting-assay,the Reed-Muench method and relative fluorescence quantitative PCR in F81/VSV system.And the results showed that the antiviral activity of rGoIFN-τagainst VSV was 4.55×105U/mg.The F81 cells were treated with different concentrations of rGoIFN-τ(55ng/mL,550ng/mL,5500ng/mL),the antiviral activity was evaluated by the Reed-Muench method and fluorescence quantitative PCR.As the results shown,after infection with 24 hours and 48 hours,the rGoIFN-τplayed effective role,which resulted in a significant decrease in the VSV virus titer and VSV-L gene transcription level relative to the virus control(VC)group(P<0.05),and the best dose of rGoIFN-τwas 5500ng/mL.I
作者
孙莹慧
夏叶
李春华
郭佳宏
蔡娟
缪德年
SUN Ying-hui;XIA Ye;LI Chun-hua;GUO Jia-hong;CAI Juan;MIAO De-nian(Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;Shouguang Agriculture and Rural Affairs Bureau,Shouguang 262700,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第4期410-415,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
上海市农业科学院卓越团队建设项目[沪农科卓越项目(2022)012]。
关键词
山羊干扰素-τ
原核表达
猫肾细胞
抗病毒活性
goat interferon-τ
prokaryotic expression
feline kidney cells
antiviral activity
