中国动物传染病学报
中國動物傳染病學報
중국동물전염병학보
CHINESE JOURNAL OF VETERINARY PARASITOLOGY
2011年
2期
8-12
,共5页
张旭%滕巧泱%闫丽萍%周洁文%徐大伟%颜丕熙%姬希文%戴晓光%王洪斌%高利%李泽君
張旭%滕巧泱%閆麗萍%週潔文%徐大偉%顏丕熙%姬希文%戴曉光%王洪斌%高利%李澤君
장욱%등교앙%염려평%주길문%서대위%안비희%희희문%대효광%왕홍빈%고리%리택군
流感病毒%NS1基因%MDCK
流感病毒%NS1基因%MDCK
류감병독%NS1기인%MDCK
Influenza A virus%NS1 protein%MDCK
根据流感病毒A/Puerto Rico/8/34株NS1基因的核苷酸序列设计引物,PCR扩增后,将NS1完整开放阅读框分别克隆于pMX载体和PET30a载体,成功构建了pMX-PR8-NS1和PET-PR8-NS1重组质粒。将PET-PR8-NS1重组质粒转化Jm109感受态大肠杆菌,诱导表达获得重组NS1蛋白免疫小鼠制备多抗血清。同时,将逆转录病毒载体系统pMX-PR8-NS1、pCI-NF-KB、PMDSV和MDSV共转染293T细胞,制备逆转录病毒样粒子。将逆转录病毒样粒子感染MDCK细胞,利用嘌呤霉素进行抗性筛选。然后经过PCR、RT-PCR、间接免疫荧光鉴定,获得稳定表达PR8病毒NS1蛋白的细胞系,将之命名为MDCK-PR8-NS1细胞系。该细胞系的建立有望为深入开展流感病毒NS蛋白生物学功能的研究以及为扩增NS1基因删除的流感病毒提供了有利的工具。
根據流感病毒A/Puerto Rico/8/34株NS1基因的覈苷痠序列設計引物,PCR擴增後,將NS1完整開放閱讀框分彆剋隆于pMX載體和PET30a載體,成功構建瞭pMX-PR8-NS1和PET-PR8-NS1重組質粒。將PET-PR8-NS1重組質粒轉化Jm109感受態大腸桿菌,誘導錶達穫得重組NS1蛋白免疫小鼠製備多抗血清。同時,將逆轉錄病毒載體繫統pMX-PR8-NS1、pCI-NF-KB、PMDSV和MDSV共轉染293T細胞,製備逆轉錄病毒樣粒子。將逆轉錄病毒樣粒子感染MDCK細胞,利用嘌呤黴素進行抗性篩選。然後經過PCR、RT-PCR、間接免疫熒光鑒定,穫得穩定錶達PR8病毒NS1蛋白的細胞繫,將之命名為MDCK-PR8-NS1細胞繫。該細胞繫的建立有望為深入開展流感病毒NS蛋白生物學功能的研究以及為擴增NS1基因刪除的流感病毒提供瞭有利的工具。
근거류감병독A/Puerto Rico/8/34주NS1기인적핵감산서렬설계인물,PCR확증후,장NS1완정개방열독광분별극륭우pMX재체화PET30a재체,성공구건료pMX-PR8-NS1화PET-PR8-NS1중조질립。장PET-PR8-NS1중조질립전화Jm109감수태대장간균,유도표체획득중조NS1단백면역소서제비다항혈청。동시,장역전록병독재체계통pMX-PR8-NS1、pCI-NF-KB、PMDSV화MDSV공전염293T세포,제비역전록병독양입자。장역전록병독양입자감염MDCK세포,이용표령매소진행항성사선。연후경과PCR、RT-PCR、간접면역형광감정,획득은정표체PR8병독NS1단백적세포계,장지명명위MDCK-PR8-NS1세포계。해세포계적건립유망위심입개전류감병독NS단백생물학공능적연구이급위확증NS1기인산제적류감병독제공료유리적공구。
In order to amplify the entire open reading frame of NS1 gene,the specific primers were designed according to the nucleotide sequence of NS gene of A/ Puerto Rico/ 8/ 34 Influenza virus were designed.The PCR product of NS1 gene was inserted into pMX vector and PET30a vector,and recombinant plasmids pMX-PR8-NS1 and PET-PR8-NS1 were generated.After being expressed in E.coli,NS1 protein was purified and injected into BALB/c mice for preparation of NS1 specific polyclonal antibody.Meanwhile,to produce retrovirus-like particles,plasmids pMX-PR8-NS1,pCI-NF-KB,PMDSV and MDSV were co-transfected into 293T cells.Then the retrovirus-like particles which produced and released into culture supernatants from 293T cell transfected were used to infect MDCK cells.The cells inserted with expected exogenous gene were selected by puromycin.After analyzed by PCR（using cell genome as template）,RT-PCR（using total cell RNA as template） and immuno-fluorescence assay,a cell line（MDCK-PR8-NS1） was obtained with can express NS1 protein stably.This cell line may provide a useful tool to study the function of NS1 gene or protein.
