中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2014年
5期
616-619
,共4页
雷立华%林群%郑辉哲%林财珠%林健清%林献忠%蔡宏达%杨庆%高友光
雷立華%林群%鄭輝哲%林財珠%林健清%林獻忠%蔡宏達%楊慶%高友光
뢰립화%림군%정휘철%림재주%림건청%림헌충%채굉체%양경%고우광
肝细胞生长因子%转染%绿色荧光蛋白质类%慢病毒属
肝細胞生長因子%轉染%綠色熒光蛋白質類%慢病毒屬
간세포생장인자%전염%록색형광단백질류%만병독속
Hepatocyte growth factor%Transfection%Green fluorescent proteins%Lentivirus
目的 构建携带增强绿色荧光蛋白(EGFP)基因的人肝细胞生长因子(HGF)基因慢病毒表达载体.方法 以pUC-SRα/HGF载体为模板,利用PCR体系扩增获得两端带有attB重组位点的HGF基因编码序列,并通过BP反应将其克隆至入门载体pDONRTM221的多克隆位点内以构建入门载体pDown-HGF.通过LR反应将pDown-HGF、pUp-EF1α、pTail-IRES/EGFP与目的载体pLV.Des3d.P/neo连接,得到慢病毒表达载体pLVneo/EF1 α-HGF-IRES-EGFP.并进行PCR鉴定和DNA测序.随后,通过脂质体将该重组载体与慢病毒包装系统共转染293FT细胞,空斑法测定病毒滴度.结果 PCR及DNA测序结果证实pLVneo/EF1α-HGF-IRES-EGFP中HGF基因目的片段插入位置和序列正确.转染pLVneo/EF1 α-HGF-IRES-EGFP后的293FT细胞在荧光显微镜下观察可见强绿色荧光.包装的慢病毒液滴度为7.9×107 TU/ml.结论 成功构建了携带EGFP基因的人HGF基因慢病毒表达载体.
目的 構建攜帶增彊綠色熒光蛋白(EGFP)基因的人肝細胞生長因子(HGF)基因慢病毒錶達載體.方法 以pUC-SRα/HGF載體為模闆,利用PCR體繫擴增穫得兩耑帶有attB重組位點的HGF基因編碼序列,併通過BP反應將其剋隆至入門載體pDONRTM221的多剋隆位點內以構建入門載體pDown-HGF.通過LR反應將pDown-HGF、pUp-EF1α、pTail-IRES/EGFP與目的載體pLV.Des3d.P/neo連接,得到慢病毒錶達載體pLVneo/EF1 α-HGF-IRES-EGFP.併進行PCR鑒定和DNA測序.隨後,通過脂質體將該重組載體與慢病毒包裝繫統共轉染293FT細胞,空斑法測定病毒滴度.結果 PCR及DNA測序結果證實pLVneo/EF1α-HGF-IRES-EGFP中HGF基因目的片段插入位置和序列正確.轉染pLVneo/EF1 α-HGF-IRES-EGFP後的293FT細胞在熒光顯微鏡下觀察可見彊綠色熒光.包裝的慢病毒液滴度為7.9×107 TU/ml.結論 成功構建瞭攜帶EGFP基因的人HGF基因慢病毒錶達載體.
목적 구건휴대증강록색형광단백(EGFP)기인적인간세포생장인자(HGF)기인만병독표체재체.방법 이pUC-SRα/HGF재체위모판,이용PCR체계확증획득량단대유attB중조위점적HGF기인편마서렬,병통과BP반응장기극륭지입문재체pDONRTM221적다극륭위점내이구건입문재체pDown-HGF.통과LR반응장pDown-HGF、pUp-EF1α、pTail-IRES/EGFP여목적재체pLV.Des3d.P/neo련접,득도만병독표체재체pLVneo/EF1 α-HGF-IRES-EGFP.병진행PCR감정화DNA측서.수후,통과지질체장해중조재체여만병독포장계통공전염293FT세포,공반법측정병독적도.결과 PCR급DNA측서결과증실pLVneo/EF1α-HGF-IRES-EGFP중HGF기인목적편단삽입위치화서렬정학.전염pLVneo/EF1 α-HGF-IRES-EGFP후적293FT세포재형광현미경하관찰가견강록색형광.포장적만병독액적도위7.9×107 TU/ml.결론 성공구건료휴대EGFP기인적인HGF기인만병독표체재체.
Objective To construct lentivirus vector expressing human hepatocyte growth factor (HGF) gene containing enhanced green fluorescent protein gene (EGFP).Methods Human HGF gene coding sequences were amplified from pUC-SRα/HGF with flanking attB sites using PCR and cloned into the multiple sites of pDONRTM221 using BP reaction for constructing the entry plasmid pDown-HGF.Then pDown-HGF,pUp-EF1α and pTail-IRES/EGFP were recombined into the target vector pLV.Des3d.P/neo by LR reaction to generate expression vector pLVneo/EF1α-HGF-IRES-EGFP.The expression vector was confirmed by PCR and DNA sequencing.293Fr cells were cotransfected with the recombinant vector and lentiviral packaging system through lipofectamine.The titer of virus was measured by plague assay.Results Both PCR and DNA sequencing analysis revealed that HGF gene was successfully inserted into the target site of pLVneo/EFlα-HGF-IRES-EGFP and the insertion sequence was correct.The green fluorescence was observed in the 293FT cells under fluorescence microscope after transfection and the titer of concentrated virus was 7.9 × 107 TU/ml.Conclusion Lentivirus vector expressing human HGF gene containing EGFP is constructed successfully.
