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
2010年
3期
232-234
,共3页
基因转染技术%表达的序列标记%逆转录聚合酶链反应%印迹法,蛋白质%防御素类
基因轉染技術%錶達的序列標記%逆轉錄聚閤酶鏈反應%印跡法,蛋白質%防禦素類
기인전염기술%표체적서렬표기%역전록취합매련반응%인적법,단백질%방어소류
Gene transfer techniques%Expresed sequence tags%Reverse transcriptase polymerase chain reaction%Blotting,western%Defensins
目的 构建大鼠β防御素2(rBD2)基因慢病毒表达载体,并转染培养细胞检测其表达,为rBD2研究及大鼠体内实验奠定基础.方法 提取大鼠上皮细胞总RNA,PCR扩增获得rBD2基因,双酶切PCR产物和慢病毒载体Lentivirus[含H1启动子和绿色荧光蛋白(GFP)],连接构成rBD2基因慢病毒表达载体LV-rBD2,行测序鉴定.用慢病毒包装系统对LV-rBD2进行慢病毒颗粒包装并梯度稀释法测定病毒滴度,病毒液感染培养细胞,RT-PCR和Western Blot检测rBD2表达.结果 凝胶电泳和测序结果表明rBD2基因克隆到慢病毒载体中,序列正确.完成慢病毒颗粒包装,调整病毒滴度至1×105ifu/μl.RT-PCR和Western-Blot显示rBD2基因获得表达.结论 rBD2基因慢病毒表达载体LV-rBD2被成功构建,能转染细胞并获得有效表达.
目的 構建大鼠β防禦素2(rBD2)基因慢病毒錶達載體,併轉染培養細胞檢測其錶達,為rBD2研究及大鼠體內實驗奠定基礎.方法 提取大鼠上皮細胞總RNA,PCR擴增穫得rBD2基因,雙酶切PCR產物和慢病毒載體Lentivirus[含H1啟動子和綠色熒光蛋白(GFP)],連接構成rBD2基因慢病毒錶達載體LV-rBD2,行測序鑒定.用慢病毒包裝繫統對LV-rBD2進行慢病毒顆粒包裝併梯度稀釋法測定病毒滴度,病毒液感染培養細胞,RT-PCR和Western Blot檢測rBD2錶達.結果 凝膠電泳和測序結果錶明rBD2基因剋隆到慢病毒載體中,序列正確.完成慢病毒顆粒包裝,調整病毒滴度至1×105ifu/μl.RT-PCR和Western-Blot顯示rBD2基因穫得錶達.結論 rBD2基因慢病毒錶達載體LV-rBD2被成功構建,能轉染細胞併穫得有效錶達.
목적 구건대서β방어소2(rBD2)기인만병독표체재체,병전염배양세포검측기표체,위rBD2연구급대서체내실험전정기출.방법 제취대서상피세포총RNA,PCR확증획득rBD2기인,쌍매절PCR산물화만병독재체Lentivirus[함H1계동자화록색형광단백(GFP)],련접구성rBD2기인만병독표체재체LV-rBD2,행측서감정.용만병독포장계통대LV-rBD2진행만병독과립포장병제도희석법측정병독적도,병독액감염배양세포,RT-PCR화Western Blot검측rBD2표체.결과 응효전영화측서결과표명rBD2기인극륭도만병독재체중,서렬정학.완성만병독과립포장,조정병독적도지1×105ifu/μl.RT-PCR화Western-Blot현시rBD2기인획득표체.결론 rBD2기인만병독표체재체LV-rBD2피성공구건,능전염세포병획득유효표체.
Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.
