中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2014年
3期
233-235
,共3页
张凤娟%唐丽%尉研%王焕琴%杨蕴芝%吴萌%张鹏%梁国栋%朱武洋
張鳳娟%唐麗%尉研%王煥琴%楊蘊芝%吳萌%張鵬%樑國棟%硃武洋
장봉연%당려%위연%왕환금%양온지%오맹%장붕%량국동%주무양
Sindbis病毒%复制子%基因
Sindbis病毒%複製子%基因
Sindbis병독%복제자%기인
Sindbis virus%Replicon%Genes
目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.
目的 構建質粒型甲病毒複製子載體繫統,併對其功能進行鑒定.方法 在XJ-160病毒感染性cDNA剋隆的基礎上,將病毒非結構基因序列分為三箇片段擴增,分步剋隆至真覈錶達載體pVAX1 CMV啟動子下遊,併用多剋隆位點序列替代病毒的結構基因構建XJ-160病毒質粒型複製子載體.將病毒覈蛋白基因及包膜糖蛋白基因分彆剋隆至pVAX1 CMV啟動子下遊構建載體的兩箇輔助質粒.通過綠色熒光蛋白報告基因及海腎熒光素酶報告基因的錶達檢驗該質粒型載體繫統的功能特性.結果 成功構建瞭包括複製子載體和輔助質粒的質粒型甲病毒複製子載體繫統,且該複製子載體繫統可成功錶達綠色熒光蛋白報告基因及海腎熒光素酶報告基因.結論 本研究構建瞭能夠錶達外源基因的質粒型甲病毒複製子載體繫統,為目標基因錶達、重組病毒顆粒製備等奠定瞭基礎.
목적 구건질립형갑병독복제자재체계통,병대기공능진행감정.방법 재XJ-160병독감염성cDNA극륭적기출상,장병독비결구기인서렬분위삼개편단확증,분보극륭지진핵표체재체pVAX1 CMV계동자하유,병용다극륭위점서렬체대병독적결구기인구건XJ-160병독질립형복제자재체.장병독핵단백기인급포막당단백기인분별극륭지pVAX1 CMV계동자하유구건재체적량개보조질립.통과록색형광단백보고기인급해신형광소매보고기인적표체검험해질립형재체계통적공능특성.결과 성공구건료포괄복제자재체화보조질립적질립형갑병독복제자재체계통,차해복제자재체계통가성공표체록색형광단백보고기인급해신형광소매보고기인.결론 본연구구건료능구표체외원기인적질립형갑병독복제자재체계통,위목표기인표체、중조병독과립제비등전정료기출.
Objective To construct a plasmid-based replicon vector system derived from Alphavirus,and its functions were identified.Methods based on XJ-160 viral infectious cDNA clone,the virus non-structural gene sequence were devided into three fragments to amplify and then cloned into the eukaryotic expression vector pVAX1 CMV promoter downstream by step clone.A multiple cloning site sequence replacing the virus structural gene,plasmid-based replicon vector derived from XJ-160 virus was constructed.Virus nucleoprotein gene and envelope glycoprotein gene were respectively cloned into pVAX1 CMV promoter downstream,constructing two helper plasmid vectors.Then by the expression of green fluorescent protein reporter gene and renilla luciferase reporter gene we test the function of the plasmid-based replicon vector system.Results The plasmid-based replicon vector system derived from XJ-160 virus was successfully constructed,and green fluorescent protein reporter gene and renilla luciferase reporter gene were successfully expressed.Conclusion We construct the replicon vector system with the independent replication and translation capabilities,and it can be used in subsequent studies,to lay the foundation of the expression of the target gene and the preparion of the recombinant viral particles.