摘要
目的应用反转录-聚合酶链反应技术克隆目的基因,构建慢病毒-血管内皮生长因子_(165)(VEGF_(165))载体,转染脂肪组织来源的干细胞(adipose tissue derived stem cells,ADSCs),并验证VEGF_(165)蛋白的体内、外表达。方法应用RT-PCR技术克隆目的基因VEGF_(165)片段,并克隆于慢病毒骨架质粒p LVX-EF1α-IRES2-Ac GFP1中,构建慢病毒载体p LVX-EF1α-VEGF_(165)-IRES2-Ac GFP1,菌落PCR及测序分析加以鉴定。慢病毒载体主体质粒p LVX-EF1α-VEGF_(165)-IRES2-Ac GFP1,辅助质粒gag-pro、vpr-pol、Tet-Off~、tat-IRES-rev和包膜质粒env(VSV-G)共转染Lenti-X 293T细胞,包装慢病毒载体并测定滴度。胶原酶消化法分离、培养ADSCs,形态学、免疫荧光及多向分化鉴定。包装后的慢病毒载体转染ADSCs,免疫荧光、ELISA鉴定VEGF_(165)在ADSCs中的体外表达。转染VEGF_(165)的ADSCs体内注射,ELISA鉴定VEGF_(165)的体内表达。结果成功克隆了目的基因VEGF_(165)片段,构建的慢病毒载体p LVX-EF1α-VEGF_(165)-IRES2-Ac GFP1经菌落PCR鉴定存在目的基因VEGF_(165)片段,测序分析证实与Genbank报道的VEGF_(165)基因序列完全一致。六质粒共转染Lenti-X 293T细胞后,荧光显微镜下可见大量绿色荧光。包装后慢病毒测定滴度为1×10~8TU·m L^(-1)。培养的ADSCs经形态学、免疫荧光及多向分化鉴定,符合文献报道的ADSCs特点。慢病毒载体转染ADSCs后经免疫荧光验证约90%ADSCs可表达VEGF_(165),同时ELISA也证明VEGF_(165)在体内、外稳定高效表达。结论经RT-PCR法克隆目的基因,成功构建了表达VEGF_(165)基因的慢病毒载体,转染ADSCs后可在体内、外稳定表达VEGF_(165)。
OBJECTIVE To clone target gene by RT-PCR method, construct VEGF165 lentivirus vectors, transfect adipose tissue derived stem cells （ADSCs） and verify the expression of VEGF165 in vitro and in vivo. METHODS RT-PCR technology was employed to clone VEGF16s gene, and this gene was cloned to lentivirus vector pLVX-EF1α-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1α-VEGFI65-IRES2-AeGFP1. Bacterial colonies PCR and sequencing analysis were used for identification. Then, Lenti-X 293T cells were transfected with main vector pLVX-EFlot-VEGF165-IRES2-AcGFP1, packaging plasmid gag-pro, vpr-pol, Tet-OffTM, tat-IRES-rev and coating plasmid env（VSV-G）. Lentiviral vectors were packaged and the titer was determined. ADSCs were isolated by collagenase digestion method, then cultured, and identificated by morphology, immunofluorescence and multi-directional differentia- tion. ADSCs was transfected with packaged VEGF16s lentivirus. Immunofluorescenee and ELISA were used to detect the expression of VEGF165 in vitro. ADSCs transfected with VEGF16s lentivirus were injected into nude mice. ELISA was used to detect the expression of VEGF16s. RESULTS The VEGF165 gene fragment was cloned successfully, and the lentiviral vector plasmid pLVX-EFlct-HVEGFx65- IRES2-AcGFP1 was confirmed to contain VEGFI65 gene fragment as shown by bacterial colonies PCR. DNA sequencing analysis con- firmed that VEGF165 gene sequencing was exactly the same with that reported by Genbank. After transfection, a large number of Lenti- X 293T cells with green fluorescence were observed by fluorescent microscopy. The concentration of the virus titer was 1× 10s TU · mL^-1. ADSCs were identified by morphology, immunofluorescenee and multi-directional differentiation methods, in line with the literature reported ADSCs characteristics. There were about 90% of ADSCs which could express VEGF165 after being transfected with the viruses by immunofluorescenee detection, also, VEGF165 protein was proved by ELISA to express stably and efficiently in vitro an
出处
《中国药学杂志》
CAS
CSCD
北大核心
2016年第3期188-196,共9页
Chinese Pharmaceutical Journal
基金
国家自然科学基金资助项目(81270548
81400677)
高等学校博士学科点专项科研基金资助项目(20110201110048)
