国际流行病学传染病学杂志
國際流行病學傳染病學雜誌
국제류행병학전염병학잡지
INTERNATIONAL JOURNAL OF EPIDEMIOLOGY AND INFECTIOUS DISEASE
2012年
3期
148-152
,共5页
房有荣%李红玉%陈科达%周文硕%阎辉
房有榮%李紅玉%陳科達%週文碩%閻輝
방유영%리홍옥%진과체%주문석%염휘
痘苗病毒%载体%构建%GFP%Zeocin
痘苗病毒%載體%構建%GFP%Zeocin
두묘병독%재체%구건%GFP%Zeocin
Vaccinia virus%Vectors%Construction%GFP%Zeocin
目的 为研究重组痘苗病毒通用载体,构建融合Zeocin和GFP双筛选标记质粒pCB-ZeoGFP.方法 质粒LeGo-G/Zeo含有Zeo-GFP融合基因片段,通过PCR反应、BamHI酶切后连接替换空白质粒pCB的筛选基因gpt,通过菌落PCR,酶切图谱分析及测序分析鉴定重组质粒,构建成功后将其与野生型痘苗病毒进行位点特异性同源重组,经Zeocin药物筛选2代后,用流式细胞仪观察重组痘苗病毒GFP表达.结果 经菌落PCR,1号、5号和10号菌落中扩增出426 bp 的目的条带,与预期的大小完全一致,经酶切分析和DNA测序进一步验证了重组质粒pCB-Zeo-GFP;该质粒与野生型痘苗病毒同源重组,得到了重组的痘苗病毒；Zeocin 筛选2代后,病毒上清感染细胞,流式细胞分析7.18％的细胞中有CFP的表达,而阴性对照仅有1.43％；Zeocin 药物筛选5代后,通过激光共聚焦可在80％以上的细胞中观察带GFP;提取的重组痘苗基因组DNA也扩增出预期大小目的条带.结论 含Zeocin和GFP双筛选标记的新型重组痘苗病毒不仅具有可观察性且具有药物抗性,非常容易进行筛选鉴定.
目的 為研究重組痘苗病毒通用載體,構建融閤Zeocin和GFP雙篩選標記質粒pCB-ZeoGFP.方法 質粒LeGo-G/Zeo含有Zeo-GFP融閤基因片段,通過PCR反應、BamHI酶切後連接替換空白質粒pCB的篩選基因gpt,通過菌落PCR,酶切圖譜分析及測序分析鑒定重組質粒,構建成功後將其與野生型痘苗病毒進行位點特異性同源重組,經Zeocin藥物篩選2代後,用流式細胞儀觀察重組痘苗病毒GFP錶達.結果 經菌落PCR,1號、5號和10號菌落中擴增齣426 bp 的目的條帶,與預期的大小完全一緻,經酶切分析和DNA測序進一步驗證瞭重組質粒pCB-Zeo-GFP;該質粒與野生型痘苗病毒同源重組,得到瞭重組的痘苗病毒；Zeocin 篩選2代後,病毒上清感染細胞,流式細胞分析7.18％的細胞中有CFP的錶達,而陰性對照僅有1.43％；Zeocin 藥物篩選5代後,通過激光共聚焦可在80％以上的細胞中觀察帶GFP;提取的重組痘苗基因組DNA也擴增齣預期大小目的條帶.結論 含Zeocin和GFP雙篩選標記的新型重組痘苗病毒不僅具有可觀察性且具有藥物抗性,非常容易進行篩選鑒定.
목적 위연구중조두묘병독통용재체,구건융합Zeocin화GFP쌍사선표기질립pCB-ZeoGFP.방법 질립LeGo-G/Zeo함유Zeo-GFP융합기인편단,통과PCR반응、BamHI매절후련접체환공백질립pCB적사선기인gpt,통과균락PCR,매절도보분석급측서분석감정중조질립,구건성공후장기여야생형두묘병독진행위점특이성동원중조,경Zeocin약물사선2대후,용류식세포의관찰중조두묘병독GFP표체.결과 경균락PCR,1호、5호화10호균락중확증출426 bp 적목적조대,여예기적대소완전일치,경매절분석화DNA측서진일보험증료중조질립pCB-Zeo-GFP;해질립여야생형두묘병독동원중조,득도료중조적두묘병독；Zeocin 사선2대후,병독상청감염세포,류식세포분석7.18％적세포중유CFP적표체,이음성대조부유1.43％；Zeocin 약물사선5대후,통과격광공취초가재80％이상적세포중관찰대GFP;제취적중조두묘기인조DNA야확증출예기대소목적조대.결론 함Zeocin화GFP쌍사선표기적신형중조두묘병독불부구유가관찰성차구유약물항성,비상용역진행사선감정.
Objective To construct the plasmid pCB-Zeo-GFP fusioned with Zeocin and GFP double selection marker,and construct recombinant vacci1nia virus to express the double selection marker.Methods plasmid LeGo-G/ Zeo which contained Zeo-GFP fusion gene fragment was amplified by PCR,digested by BamH I enzyme,and then ligated with the blank plasmid pCB treated with the same enzyme to replace the original marker gene gpt.Through colony PCR,the recombinant plasmid pCB-Zeo-GFP was identified by restriction map analysis and sequencing analysis,the recombinant vaccinia virus was constructed through homologous recombination at homology-bit point TK area.After Zeocin screening of two generations,GFP expression of the cells infected by the viral supematant was analyzed with flow cytometry.Results The target 426 bp band was amplified on the 1st,the 5th and the 10th colonies by colony PCR,and was fully consistent with the expected size; the recombinant plasmid pCB-Zeo-GFP was verified by restriction enzyme analysisand DNA sequencing,The recombinant vaccinia virus was constructed through homologous recombination and Zeocin screening of the two generations,GFP expression of the cells infected with the viral supematant was up to 7.18％ by flow cytometry while the control was up to 1.43 ％.Through Zeocin screening of the five generations,GFP was observed on more than 80％ cells by laser confocal and the target band was amplified from recombinant vaceinia virus genomic DNA.Conclusions The recombinant vaccinia virus is not only observable but also resistant to drug resistance,so it is easy to be screened and identified.
