CAJ | 학술논문

目的利用慢病毒载体系统包装含人类免疫缺陷病毒1型(HIV-1)病毒蛋白R(Vpr)的重组慢病毒,使之感染原发性渗出性淋巴瘤(PEL)细胞BCBL-1,并检测Vpr蛋白对细胞中卡波济肉瘤相关疱疹病毒(KSHV)潜伏感染与裂解性复制的影响.方法从实验室先前构建的重组真核表达质粒pCI-neo-Vpr中扩增出Vpr基因,插入到pHAGE-CMV-MCS-IzsGreen载体中构建成重组慢病毒质粒pHAGE-Vpr,利用脂质体将其与包装质粒psPAX2及包膜质粒pMD2.G共转染293T细胞,荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)的表达,293T细胞培养上清经0.45 μm滤器过滤后即获得重组病毒悬液.慢病毒系列稀释后感染293T细胞,荧光计数法测定病毒滴度,逆转录PCR(RT-PCR)检测Vpr基因在293T细胞中的转录.以感染复数(MOI)为1的病毒量感染靶细胞BCBL-1,荧光显微镜观察靶细胞中GFP的表达,并利用RT-PCR和和免疫印迹(Western blot)技术分别检测Vpr基因在靶细胞中的转录和表达情况,同时检测KSHV裂解期基因复制与转录激活子(Rta)mRNA转录及蛋白表达.结果经酶切鉴定、核酸序列测定和293T细胞中GFP表达证实成功包装了携带HIV-1Vpr基因的重组慢病毒,滴度为4×107TU/ml.重组慢病毒感染BCBL-1细胞后,细胞中有GFP表达,RT-PCR和Western blot均能够在相应位置检测到目的基因Vpr的条带,并且Vpr降低了KSHV RtamRNA转录及蛋白表达水平.结论重组慢病毒介导的HIV-1 Vpr蛋白过表达能够抑制KSHV裂解性复制、增强病毒的潜伏感染.
목적 이용만병독재체계통포장함인류면역결함병독1형(HIV-1)병독단백R(Vpr)적중조만병독,사지감염원발성삼출성림파류(PEL)세포BCBL-1,병검측Vpr단백대세포중잡파제육류상관포진병독(KSHV)잠복감염여렬해성복제적영향.방법 종실험실선전구건적중조진핵표체질립pCI-neo-Vpr중확증출Vpr기인,삽입도pHAGE-CMV-MCS-IzsGreen재체중구건성중조만병독질립pHAGE-Vpr,이용지질체장기여포장질립psPAX2급포막질립pMD2.G공전염293T세포,형광현미경관찰293T세포중록색형광단백(GFP)적표체,293T세포배양상청경0.45 μm려기과려후즉획득중조병독현액.만병독계렬희석후감염293T세포,형광계수법측정병독적도,역전록PCR(RT-PCR)검측Vpr기인재293T세포중적전록.이감염복수(MOI)위1적병독량감염파세포BCBL-1,형광현미경관찰파세포중GFP적표체,병이용RT-PCR화화면역인적(Western blot)기술분별검측Vpr기인재파세포중적전록화표체정황,동시검측KSHV렬해기기인복제여전록격활자(Rta)mRNA전록급단백표체.결과 경매절감정、핵산서렬측정화293T세포중GFP표체증실성공포장료휴대HIV-1Vpr기인적중조만병독,적도위4×107TU/ml.중조만병독감염BCBL-1세포후,세포중유GFP표체,RT-PCR화Western blot균능구재상응위치검측도목적 기인Vpr적조대,병차Vpr강저료KSHV RtamRNA전록급단백표체수평.결론 중조만병독개도적HIV-1 Vpr단백과표체능구억제KSHV렬해성복제、증강병독적잠복감염.
Objective To package the recombinant lentivirus containing HIV-1 Vpr gene and detect the effect of Vpr protein expression on the latent infection and lyric replication of KSHV.Methods The fragment of Vpr gene from expression plasmid pCI-neo-Vpr was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant plasmid pHAGE-Vpr,package vector psPAX2 and envelope vector pMD2.G were cotransfected into 293T cells.GFP expression was observed by fluorescent microscopy.Culture media of 293T cells were harvested and filtered through 0.45 μm filter.After 293T cells were infected by a series of diluted lentivirus,the virus titer was checked by observing GFP expression.Vpr mRNA transcripts in 293T cells were detected by RT-PCR.Then BCBL-1 cells were infected by the recombinant lentivirus with 1 MOI,GFP expression was observed by fluorescent microscopy,and the mRNA transcripts and protein expression of Vpr in BCBL-1 cells were detected by RT-PCR and Western blot.Meanwhile,the mRNA transcripts and protein expression of KSHV lytic cycle gene Rta were detected by RT-PCR and Western blot,respectively.Results The recombinant lentivirus carrying HIV-1 Vpr was packaged successfully with the virus titer of 4 × 107 TU/ml.After infected with lentivirus,BCBL-1 cells could express GFP,and the exact band of Vpr was detectable by RT-PCR and Western blot.Moreover,the expression of KSHV Rta mRNA and protein were downregulated by Vpr protein.Conclusion Overexpression of HIV-1 Vpr mediated by the recombinant lentivirus could inhibit KSHV lytic replication and enhance KSHV latent infection.