CAJ | 학술논문

目的 NS1蛋白在流感病毒复制和传播中起到重要的调节作用,其功能多样,如能调节宿主细胞的蛋白合成、诱导感染细胞的凋亡以及拮抗IFN作用等,此外还有许多功能至今尚未明了.因此,研制一个特异性高的抗NS1抗体,对NS1蛋白功能的进一步研究起到重要作用.方法 RT-PCR扩增流感病毒NS1基因,将其克隆进原核表达载体pET-28a(+)中.然后在大肠埃希菌BL21中表达,用切胶纯化方法获取NS1融合蛋白.用该蛋白免疫新西兰大白兔,制备效价较高的多克隆抗体,并测定其效价(间接ELISA法).用病毒感染细胞实验、Western印迹法和间接免疫荧光实验等方法,观察病毒复制过程中NS1蛋白的表达及细胞定位情况.结果首先构建了能表达NS1蛋白的质粒,并经原核细胞表达获得了NS1蛋白.然后用该蛋白免疫大白兔,获得了效价较高(1∶256 000)的抗NS1蛋白抗体.实验发现,该抗体可以特异性结合非依赖亚型的甲型流感病毒NS1蛋白.病毒感染后6h即可以检测到NS1蛋白,随着病毒复制时间的延长NS1蛋白表达量变化不明显；24h后NS1蛋白主要分布于细胞核内并能定位于核仁中,也有部分位于细胞质.结论本研究通过原核表达获得了NS1蛋白,并利用常规的免疫方法获得了效价较高的抗NS1多克隆抗体.所制备的多克隆抗体能够有效定位NS1蛋白的表达,并且能和甲型流感病毒各亚型结合,具有较高的特异性.该抗体不仅能用于NS1蛋白功能及其对宿主细胞影响的研究,而且可用于鉴别诊断猪或其他动物中的病毒感染还是接种疫苗后的反应,有较好的开发应用前景.
목적 NS1단백재류감병독복제화전파중기도중요적조절작용,기공능다양,여능조절숙주세포적단백합성、유도감염세포적조망이급길항IFN작용등,차외환유허다공능지금상미명료.인차,연제일개특이성고적항NS1항체,대NS1단백공능적진일보연구기도중요작용.방법 RT-PCR확증류감병독NS1기인,장기극륭진원핵표체재체pET-28a(+)중.연후재대장애희균BL21중표체,용절효순화방법획취NS1융합단백.용해단백면역신서란대백토,제비효개교고적다극륭항체,병측정기효개(간접ELISA법).용병독감염세포실험、Western인적법화간접면역형광실험등방법,관찰병독복제과정중NS1단백적표체급세포정위정황.결과 수선구건료능표체NS1단백적질립,병경원핵세포표체획득료NS1단백.연후용해단백면역대백토,획득료효개교고(1∶256 000)적항NS1단백항체.실험발현,해항체가이특이성결합비의뢰아형적갑형류감병독NS1단백.병독감염후6h즉가이검측도NS1단백,수착병독복제시간적연장NS1단백표체량변화불명현；24h후NS1단백주요분포우세포핵내병능정위우핵인중,야유부분위우세포질.결론 본연구통과원핵표체획득료NS1단백,병이용상규적면역방법획득료효개교고적항NS1다극륭항체.소제비적다극륭항체능구유효정위NS1단백적표체,병차능화갑형류감병독각아형결합,구유교고적특이성.해항체불부능용우NS1단백공능급기대숙주세포영향적연구,이차가용우감별진단저혹기타동물중적병독감염환시접충역묘후적반응,유교호적개발응용전경.
Objective NS1 protein of influenza virus plays an important regulatory role on the replication and dissemination.It has many functions,such as regulating host cell protein synthesis,inducing apoptosis of infection cell,as well as antagonistic action of interferon,but many functions are still unclear so far.Therefore,the development of a highly specific anti-NS1 antibody plays an important role in NS1 protein for further functional studies.Methods A NS1 gene of influenza virus was amplified by RT-PCR.A plasmid (pET-28a-NS1) was constructed from the expression vector pET-28a( + ).Then NS1 fusion protein was expressed in the prokaryotic cell ( E.coli BL21 ).The purified NS1 protein immunized New Zealand big white rabbit.The specificity of antibody serum was detected by ELISA.The bio-characteristics of the antibody were determined with virus infected cell experiment,Western blotting method and indirect immune-fluorescence test.Results A plasmid (pET-28a-NS1) which can express of NS1 protein was constructed.Then,the NS1 protein by prokaryotic expression was used to immunize rabbits.The anti-NS1 antibody which had a specificity of 1∶256000 was obtained.Experiments showed that the antibody could bind with NS1 protein of influenza A virus independence on subtypes.Therefore,it could detect the expression and location of NS1 protein in the infected cells.For example,NS1 protein could be detected after infected for 6 hours,and did not change significantly with the time of virus replication.After 24 hours,NS1 protein was mainly distributed in the cell nucleus and could locate in the nucleolus,moreover also had a portion located in the cytoplasm.Conclusions In this study,the purified NS1 protein are obtained through prokaryotic expression and higher specificity of polyclonal antibody against NS1 using routine immunization method.The antibody can bind specifically with NS1 proteins of influenza A viruscs independent its subtypes,and can be used to determine the NS1 protein expression effectively in the infected cells.The antibody can be used not only for researching NS1 protein function and its effects on host cells,but also differential diagnosis of viral infection or vaccination of poultry pig and other animals,have good development and application prospect.