CAJ | 학술논문

背景:骨形态发生蛋白是一种具有潜在活性的蛋白质,当骨组织损伤时其迅速增加并活性的增强,与载体复合能修复动物骨缺损,但将其用做基因治疗的研究未见报道.目的:构建表达重组人骨形态发生蛋白2基因的重组反转录病毒载体,探讨其在成骨细胞中的生物学作用.方法:根据Genbank中人骨形态发生蛋白2基因序列设计并合成骨形态发生蛋白2特异性引物,高保真PCR扩增骨形态发生蛋白2基因,同源重组法将骨形态发生蛋白2 PCR片段连接与克隆载体pDNR-CMV,构成pDNR-CMV-BMP2,经酶切、PCR和测序鉴定后,将重组质粒pDNR-CMV-BMP2和反转录病毒空质粒pLP-LNCX以loxP位点进行同源重组,构成反转录病毒载体pLP-LNCX-BMP2,转染入包装细胞PT67进行病毒包装,并用NIH3T3细胞进行病毒滴度测定;将反转录病毒感染人成骨细胞,四甲基偶氮唑盐法检测细胞生长变化,转染48 h后Western blotting检测骨形态发生蛋白2蛋白表达.结果与结论:pDNR-CMV-BMP2质粒Sall和EcoRI双酶切、PCR及测序结果均正确,重组质粒pLP-LNCX-BMP2经氯霉素及蔗糖筛选得到的阳性克隆骨形态发生蛋白2 PCR结果阳性,酶切产物与预期相一致;病毒载体pLP-LNCX-BMP2转染PT67后,G418筛选可得到稳定细胞克隆,其上清液中病毒滴度可达到5×10~8pfu;四甲基偶氮唑盐检测中反转录病毒组与正常对照组相比,72 h细胞抑制率无明显差别(P＞0.05),转染48 h后Western blotting可见骨形态发生蛋白2蛋白高表达.结果说明,实验成功克隆了骨形态发生蛋白2基因并构建其反转录病毒表达载体.
배경:골형태발생단백시일충구유잠재활성적단백질,당골조직손상시기신속증가병활성적증강,여재체복합능수복동물골결손,단장기용주기인치료적연구미견보도.목적:구건표체중조인골형태발생단백2기인적중조반전록병독재체,탐토기재성골세포중적생물학작용.방법:근거Genbank중인골형태발생단백2기인서렬설계병합성골형태발생단백2특이성인물,고보진PCR확증골형태발생단백2기인,동원중조법장골형태발생단백2 PCR편단련접여극륭재체pDNR-CMV,구성pDNR-CMV-BMP2,경매절、PCR화측서감정후,장중조질립pDNR-CMV-BMP2화반전록병독공질립pLP-LNCX이loxP위점진행동원중조,구성반전록병독재체pLP-LNCX-BMP2,전염입포장세포PT67진행병독포장,병용NIH3T3세포진행병독적도측정;장반전록병독감염인성골세포,사갑기우담서염법검측세포생장변화,전염48 h후Western blotting검측골형태발생단백2단백표체.결과여결론:pDNR-CMV-BMP2질립Sall화EcoRI쌍매절、PCR급측서결과균정학,중조질립pLP-LNCX-BMP2경록매소급자당사선득도적양성극륭골형태발생단백2 PCR결과양성,매절산물여예기상일치;병독재체pLP-LNCX-BMP2전염PT67후,G418사선가득도은정세포극륭,기상청액중병독적도가체도5×10~8pfu;사갑기우담서염검측중반전록병독조여정상대조조상비,72 h세포억제솔무명현차별(P＞0.05),전염48 h후Western blotting가견골형태발생단백2단백고표체.결과설명,실험성공극륭료골형태발생단백2기인병구건기반전록병독표체재체.
BACKGROUND: Bone morphogenetic protein (BMP) is a protein possesses potential activity, which can increase and enhance its activity when the bone issues are damaged, so it can be used to repair the bone defects when combined with carrier. However,there are few reports concerning it as gene therapy.OBJECTIVE: To construct recombinant retroviral vector expressing human BMP2 gene and to discuss its biological function in ostecblasts.METHODS: BMP2-specific primers were designed and synthesized according gene sequence of human BMP2 gene in Genbank, then BMP2 gena was amplified by Hifi PCR, which was recombined with cloning vector pDNR-CMV homologousiy into pDNR-CMV-BMP2 plasmid identified by BMP2 PCR and enzyme digestion of Sail and EcoRI as well as gene sequencing; recombinant plasmid pDNR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombined homologously in IoxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67 and the supernatant was collected to assay viral titre. Human osteoblast was infected with retrovirus, then the growth of cells were observed by MTT, and the expression of BMP2 protein was detected by Western blotting at 48 hours transfectionRESULTS AND CONCLUSION: Digestion, BMP2 PCR and gene sequencing of pDNR-CMV-BMP2 were coincided with expected. After transfected with plasmid pLP-LNCX-BMP2, PT67 cells could be screened with G418 only to get stably integrated in BMP2, of whose supemanant viral titra amounted to 5×10~8 pfu/mL. MTT assay showed that there had no evident difference in cellular inhibition between the normal and retrovirus groups at 72 hours after transfection (P ＞ 0.05); Western blotting showed that the BMP2 was strong expressed at 48 hours after transfection. It demonstrated that BMP2 gene was successful cloned and its retrovirus vector was constructed.