摘要
为获得增强型猫ω干扰素(FeIFN-ω),本研究通过分子对接方法预测和参考相关位点研究,设计3组定点突变引物。以pJET-FeIFN-ω为模板分别扩增FeIFN-ω基因及其3个突变基因(FeIFN-ω-R35L、FeIFN-ω-A156R、FeIFN-ω-T188R),并连接慢病毒载体pLVX-IRSE,构建重组质粒pLVX-FeIFN-ω、pLVX-FeIFN-ω-R35L、pLVX-FeIFN-ω-A156R、p LVX-FeIFN-ω-T188R。4个重组质粒分别与包装质粒psPAX2和膜蛋白质粒pMD2.G共转染293T细胞包装慢病毒,48 h后收获细胞上清即为慢病毒。分别将4个慢病毒转导293悬浮细胞(293s),扩大培养后收获细胞上清,经镍离子亲和层析柱纯化蛋白,并经SDS-PAGE检测、BCA法测蛋白浓度。结果显示,rFeIFN-ω、rFeIFN-ω-R35L、rFeIFN-ω-A156R、rFeIFN-ω-T188R在293s细胞中得到高效表达,且蛋白纯化效果好;经BCA法测浓度,4种重组蛋白浓度均约为200μg/mL,即获得了重组干扰素rFeIFN-ω、rFeIFN-ω-R35L、rFeIFN-ω-A156R、rFeIFN-ω-T188R。4种重组干扰素分别经猫肾细胞(CRFK)孵育18 h后接种猫杯状病毒(FCV)或猫疱疹病毒(FHV)12 h,经相对荧光定量PCR法检测干扰素抗FCV和FHV活性;4种重组干扰素分别经CRFK细胞孵育12 h,经相对荧光定量PCR法检测干扰素刺激基因ISG15、Viperin、IFITM1的转录水平,结果显示:实验组与空白对照组差异极显著(P<0.01),实验组中rFeIFN-ω-R35L、rFeIFN-ω-A156R与rFeIFN-ω均差异不显著(P>0.05)或表现为活性降低,rFeIFN-ω-T188R与rFeIFN-ω、rFeIFN-ω-R35L、rFeIFN-ω-A156R差异均显著(P<0.05)表现为活性增强,并且r FeIFN-ω-T188R抗病毒活性及诱导ISGs的转录水平均比rFeIFN-ω高1倍左右。选择r FeIFN-ω和rFeIFN-ω-T188R分别与His纯化树脂结合,加入等量表达干扰素受体(IFNAR1/R2)的重组质粒转染293T细胞的裂解液,4℃结合过夜,经western blot鉴定干扰素与IFNAR1/R2结合亲和力分析,结果显示,rFeIFN-ω-T188R与IFNAR1的结合能力比r FeIFN-ω高1倍左右,且两者与IFNAR2的结合能力差异不�
In order to obtain enhanced feline interferon(FeIFN-ω),three groups of site-directed mutagenesis primers were designed by molecular docking method prediction and reference to related sites study.The FeIFN-ωgene and the three mutant genes(FeIFN-ω-R35L,FeIFN-ω-A156 R,FeIFN-ω-T188R)were amplified from pJET-FeIFN-ωtemplate and cloned into pLVXIRSE vector.The recombinant plasmids pLVX-FeIFN-ω,pLVX-FeIFN-ω-R35L,p LVX-FeIFN-ω-A156 R and p LVX-FeIFN-ω-T188R were constructed and identified by double enzyme digestion.Next,293 T cells were co-transfected with each of the four recombinant plasmids with the packaging plasmid pSPAX2 and the membrane protein plasmid p MD2.G to package lentivirus,and the cell supernatant was collected at 48 hours.The recombinant lentiviruses were transduced into 293 suspension(293 s)cells,and the recombinant proteins were purified from the supernatant of transduced 293 s cells by Nickel particle affinity chromatography column and determined by SDS-PAGE.BCA method was used to measure the protein concentration.The results showed that rFeIFN-ω,rFeIFN-ω-R35L,rFeIFN-ω-A156 R and r FeIFN-ω-T188R were highly expressed in 293 s cells and the purification effect was good;In addition,the concentration of the four recombinant proteins were about 200μg/mL detected by BCA method.These results suggest that the recombinant interferons r FeIFN-ω,rFeIFN-ω-R35L,rFeIFN-ω-A156 R and rFeIFN-ω-T188R are obtained.Furthermore,the feline renal cells(CRFK)were incubated with four interferons separately for 18 hours,then inoculated with feline calicivirus(FCV)or feline herpes virus(FHV)for additional 12 hours.The anti-FCV and FHV activities of 4 recombinant interferons were detected by relative fluorescence quantitative PCR.Besides,the transcription levels of interferon stimulated genes ISG15,Viperin and IFITM1 in CRFK cells were measured by relative fluorescence quantitative PCR at 12 hours post recombinant interferons treatment.The results showed that the anti-virus activity in the experimental group
作者
李茵
潘玉迪
田进
曲连东
LI Yin;PAN Yu-di;TIAN Jin;QU Lian-dong(Veterinary Public Health Key Laboratory of the Ministry of Agricultural,State Key Laboratory of Veterinary Biotechnology,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第2期195-201,213,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然基金面上项目(31770172)。
关键词
猫ω干扰素
真核表达
生物活性
FeIFN-ω
eukaryotic expression
biological activity
