摘要
背景:内部核糖体进入位点序列载体能将上下游基因共同转录,故通过此载体可使连接在一起的人骨形态发生蛋白2和人成纤维细胞生长因子2同时高表达,可为骨缺损的治疗提供新方法。目的:实验通过引入内部核糖体进入位点序列,采用基于attLXattR的λ噬茵体位点特异性重组系统的GatewayTM技术构建带有人骨形态发生蛋白2和人成纤维细胞生长因子2双基因重组腺病毒载体并进行鉴定。方法:用聚合酶链反应方法扩增人骨形态发生蛋白2和人成纤维细胞生长因子2目的基因,将两个基因亚克隆至pIRES2-EGFP载体中,构建phBMP2-IRES-hFGF2载体,通过BP反应将hBMP2-IRES-hFGF2重组到载体pDONR221中,再通过LR反应将其转移到腺病毒载体pAd/CMV/V5-DEST中,线性化后转染293细胞进行包装获得重组腺病毒Ad-hBMP2-IRES-hFGF2。结果与结论:phBMP2-IRES-hFGF2经酶切及测序鉴定正确,带hBMP2-IRES-hFGF2的载体在293细胞中包装成功,获得成熟的病毒颗粒,病毒滴度为2.82×1010ifu/mL,证实成功构建了带有人骨形态发生蛋白2和人成纤维细胞生长因子2双基因重组腺病毒载体。
BACKGROUND：The upstream and downstream genes can be co-transcribed by internal ribosome entry site（IRES） sequence,by which human bone morphogenetic protein 2（hBMP2） and human fibroblast growth factor 2（hFGF2） genes can be highly co-expressed,thus,it provides a new way for repairing bone defects.OBJECTIVE：To construct the recombinant adenovirus vector co-expressing hBMP2 and hFGF2 depending on IRES sequence by λ phage-site specific recombination systems and to verify the correct recombination.METHODS：BMP-2cDNA and FGF-2 cDNA were amplified by PCR,and were subcloned into the pIRES2-EGFP plasmid to obtain phBMP2-IRES-hFGF2 plasmid.It was subcloned into entry vector pDONR221 by BP reaction and subcloned into pAd/CMV/V5-DEST vector by LR reaction and linearization transfection 293 cells to obtain pAd-hBMP2-IRES-hFGF2.RESULTS AND CONCLUSION：The target gene of hBMP2-IRES-hFGF2 was transferred into AD/CMV/V5-DEST vector correctly and it was confirmed by sequencing.The expression clone pAd-hBMP2-IRES-hFGF2 was packaged into maturated adenovirus successfully;the titer of Ad-hBMP2-IRES-hFGF2 was 2.82×1010 ifu/mL.The results demonstrated that adenovirus vector co-expressing hBMP2 and hFGF2 are constructed successfully.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第24期4402-4406,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
广东省自然科学基金资助项目(8452402301001081)~~
