中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2010年
5期
453-456
,共4页
β防御素2%转染%慢病毒属%RNA干扰%沉默效应
β防禦素2%轉染%慢病毒屬%RNA榦擾%沉默效應
β방어소2%전염%만병독속%RNA간우%침묵효응
β-defensin-2 (rBD2)%Transfection%Lentivirus%RNA interferenc%Silencing effect
目的 构建大鼠β防御素2(rBD2)基因RNAi慢病毒重组载体,转染培养细胞,检测其沉默效应,筛选出最佳的RNAi慢病毒载体.方法 序列软件设计3条针对rBD2基因CDS区的siRNA序列,合成单链后退火形成双链DNA,分别与酶切处理的慢病毒载体lentivirus连接构成3个RNAi慢病毒重组载体,再转化细菌,测序鉴定.脂质体法转染细胞,荧光实时定量PCR(RT-PCR)及Western免疫印迹测rBD-2 mRNA及蛋白表达,筛选沉默效果最佳的为LV-shrBD2载体.用慢病毒包装系统对LV-shrBD2进行慢病毒颗粒包装并梯度稀释法测定病毒滴度.结果 凝胶电泳显示3个RNAi慢病毒重组载体的PCR产物为316 bp,测序结果表明序列正确.转染细胞后RT-PCR及Western免疫印迹检测,siRNA序列1构建的重组载体mRNA抑制率达82%,干扰效率最高,为所需的rBD2基因RNAi慢病毒载体LV-shrBD2.包装慢病毒颗粒并调整病毒滴度至1×105ifu/μl.结论 成功构建并筛选出沉默效应最佳的rBD2基因RNAi慢病毒表达载体LV-sh1rBD2,为进一步开展rBD2研究提供了依据.
目的 構建大鼠β防禦素2(rBD2)基因RNAi慢病毒重組載體,轉染培養細胞,檢測其沉默效應,篩選齣最佳的RNAi慢病毒載體.方法 序列軟件設計3條針對rBD2基因CDS區的siRNA序列,閤成單鏈後退火形成雙鏈DNA,分彆與酶切處理的慢病毒載體lentivirus連接構成3箇RNAi慢病毒重組載體,再轉化細菌,測序鑒定.脂質體法轉染細胞,熒光實時定量PCR(RT-PCR)及Western免疫印跡測rBD-2 mRNA及蛋白錶達,篩選沉默效果最佳的為LV-shrBD2載體.用慢病毒包裝繫統對LV-shrBD2進行慢病毒顆粒包裝併梯度稀釋法測定病毒滴度.結果 凝膠電泳顯示3箇RNAi慢病毒重組載體的PCR產物為316 bp,測序結果錶明序列正確.轉染細胞後RT-PCR及Western免疫印跡檢測,siRNA序列1構建的重組載體mRNA抑製率達82%,榦擾效率最高,為所需的rBD2基因RNAi慢病毒載體LV-shrBD2.包裝慢病毒顆粒併調整病毒滴度至1×105ifu/μl.結論 成功構建併篩選齣沉默效應最佳的rBD2基因RNAi慢病毒錶達載體LV-sh1rBD2,為進一步開展rBD2研究提供瞭依據.
목적 구건대서β방어소2(rBD2)기인RNAi만병독중조재체,전염배양세포,검측기침묵효응,사선출최가적RNAi만병독재체.방법 서렬연건설계3조침대rBD2기인CDS구적siRNA서렬,합성단련후퇴화형성쌍련DNA,분별여매절처리적만병독재체lentivirus련접구성3개RNAi만병독중조재체,재전화세균,측서감정.지질체법전염세포,형광실시정량PCR(RT-PCR)급Western면역인적측rBD-2 mRNA급단백표체,사선침묵효과최가적위LV-shrBD2재체.용만병독포장계통대LV-shrBD2진행만병독과립포장병제도희석법측정병독적도.결과 응효전영현시3개RNAi만병독중조재체적PCR산물위316 bp,측서결과표명서렬정학.전염세포후RT-PCR급Western면역인적검측,siRNA서렬1구건적중조재체mRNA억제솔체82%,간우효솔최고,위소수적rBD2기인RNAi만병독재체LV-shrBD2.포장만병독과립병조정병독적도지1×105ifu/μl.결론 성공구건병사선출침묵효응최가적rBD2기인RNAi만병독표체재체LV-sh1rBD2,위진일보개전rBD2연구제공료의거.
Objective To construct RNAi lentiviral recombinant vector of rat β-defensin-2 (rBD2) gene, detect its silencing effect by transfecting cultured cells, and to identify the RNAi lentiviral vector with best silencing effects. Methods Three siRNA sequences binding to CDS region of rBD2 gene were designed with software and used to generate single stands which formed double-stranded DNA by annealing. Three RNAi lentiviral recombinant vectors were constructed by connecting these DNAs to enzyme- digested lentivirus vectors and were identified by sequencing after transfecting into bacteria. Recombinant vector was transfected into cells by liposome. Expressions of rBD2 mRNA and protein were tested with fluorescence RTPCR and Western blotting. Recombinant vector with best silencing effect was screened as LV-shrBD2. The virus- like particles of LV- shrBD2 was packed with lentiviral packaging system and the viral titer was determined by slow-gradient dilution. Results Gel electrophoresis showed that the PCR products of three siRNAs were 316 bp in size with correct sequences. RT-PCR and Western blotting indicated that the recombinant vector with siRNA sequence 1 exhibited the best interference effect with an inhibition rate of 82%, and was therefore identified as rBD2 gene RNAi lentiviral vector LV-shrBD2. The lentiviral vector particle packaging was complete, and the virus titer was adjusted to 1×105 ifu/μl. Conclusion The RNAi lentiviral expression vector of rBD2 gene LV-shrBD2 that exhibits best gene-silencing effect is successfully constructed, which may provide evidences for further research of rBD2.
