国际生物制品学杂志
國際生物製品學雜誌
국제생물제품학잡지
INTERNATIONAL JOURNAL OF BIOLOGICALS
2013年
6期
296-299
,共4页
呼吸道合胞病毒%质粒%病毒结构蛋白质类
呼吸道閤胞病毒%質粒%病毒結構蛋白質類
호흡도합포병독%질립%병독결구단백질류
Respiratory syncytial virus%Plasmids%Viral structural proteins
目的 构建可用于表达呼吸道合胞病毒(respiratory syncytial virus,RSV)反向遗传操作中所需的4个功能性结构蛋白的辅助质粒.方法 利用RT-PCR扩增RSV Long株的核蛋白(N)、磷蛋白(P)、大蛋白(L)及转录延长/终止抑制因子M2-1基因.将N、P、M2-1基因PCR产物双酶切后连接表达载体pCI;L基因分两段扩增并分别与pMD 19-T simple载体连接,再先后切下与pCI连接.将构建得到的4个辅助质粒测序并转染Veto细胞,通过间接免疫荧光法检测相应蛋白的表达.结果 扩增得到N、P、M2-1和L4个结构蛋白基因,相应构建的辅助质粒经序列测定,与GenBank公布的RSV Long株序列完全一致;间接免疫荧光法检测表明,N、P、M2-1能在Vero细胞中表达.结论 在分子水平构建了RSV反向遗传学研究中所需的4个辅助质粒,并成功表达出3个结构蛋白.
目的 構建可用于錶達呼吸道閤胞病毒(respiratory syncytial virus,RSV)反嚮遺傳操作中所需的4箇功能性結構蛋白的輔助質粒.方法 利用RT-PCR擴增RSV Long株的覈蛋白(N)、燐蛋白(P)、大蛋白(L)及轉錄延長/終止抑製因子M2-1基因.將N、P、M2-1基因PCR產物雙酶切後連接錶達載體pCI;L基因分兩段擴增併分彆與pMD 19-T simple載體連接,再先後切下與pCI連接.將構建得到的4箇輔助質粒測序併轉染Veto細胞,通過間接免疫熒光法檢測相應蛋白的錶達.結果 擴增得到N、P、M2-1和L4箇結構蛋白基因,相應構建的輔助質粒經序列測定,與GenBank公佈的RSV Long株序列完全一緻;間接免疫熒光法檢測錶明,N、P、M2-1能在Vero細胞中錶達.結論 在分子水平構建瞭RSV反嚮遺傳學研究中所需的4箇輔助質粒,併成功錶達齣3箇結構蛋白.
목적 구건가용우표체호흡도합포병독(respiratory syncytial virus,RSV)반향유전조작중소수적4개공능성결구단백적보조질립.방법 이용RT-PCR확증RSV Long주적핵단백(N)、린단백(P)、대단백(L)급전록연장/종지억제인자M2-1기인.장N、P、M2-1기인PCR산물쌍매절후련접표체재체pCI;L기인분량단확증병분별여pMD 19-T simple재체련접,재선후절하여pCI련접.장구건득도적4개보조질립측서병전염Veto세포,통과간접면역형광법검측상응단백적표체.결과 확증득도N、P、M2-1화L4개결구단백기인,상응구건적보조질립경서렬측정,여GenBank공포적RSV Long주서렬완전일치;간접면역형광법검측표명,N、P、M2-1능재Vero세포중표체.결론 재분자수평구건료RSV반향유전학연구중소수적4개보조질립,병성공표체출3개결구단백.
Objective To construct helper plasmids expressing nucleoprotein (N),phosphoprotein (P),large protein (L) and transcription elongation/anti-termination factor M2-1 of respiratory syncytial virus (RSV).Methods N,P,M2-1 genes were amplified by RT-PCR technique.The PCR products were cloned into pCI vectors.L gene was amplified as two fragments which were then cloned into pCI vector,respectively,with help of the intermediate vector pMD 19-T simple.The resultant helper plasmids (pCI-N,pCI-P,pCI-L,pCI-M2-1) were identified by sequencing analysis and transfected into Vero cells.The expressed proteins were detected by indirect immunofluorescence assay (IFA).Results Four plasmids encoding N,P,L and M2-1 were constructed as demonstrated by sequencing analysis and sequence alignment with RSV Long strain in GenBank.N,P,M2-1 proteins were expressed in Vero cells as confirmed by IFA.Conelusion Four helper plasmids are constructed genetically and three structural proteins are successfully expressed.
