人ALPL基因慢病毒载体的构建及鉴定

Construction and identification of human recombinant lentiviraI vector of ALPL gene

目的:构建肝/骨/肾型碱性磷酸酯酶(alkaline phosphatase,liver/bone/kidney,ALPL)基因慢病毒表达载体并观察其在骨髓间充质干细胞(BMMSC)中的表达情况,为进一步研究ALPL基因功能提供基础工具。方法:将PCR扩增的人ALPL基因片段和pENTR-2B入门质粒载体的双酶切产物进行连接后,转化感受态细胞;再将所得的pENTR-2B-ALPL入门质粒与Plenti6.3/V5-DEST载体进行重组,并经DNA测序鉴定后,转染GP2-293T细胞,制备慢病毒颗粒;最后用含Plenti6.3-ALPL表达载体的病毒感染BMMSC,并通过ALP染色、RT-PCR、Western blot检测ALPL基因和组织非特异性碱性磷酸酶(tissue-nonspecific alkaline phosphatase,TNAP)的表达。结果:PCR和测序证实人ALPL基因正确插入慢病毒载体;ALP染色、RT-PCR、Western blot检测结果显示,人ALPL慢病毒过表达质粒及相应病毒颗粒能在BMMSC中有效过表达ALPL基因。结论:成功建立ALPL慢病毒表达系统,并为后期深入了解TNAP功能打下了基础。

AIM： To construct human recombinant lentiviral vector of alkaline phosphatase liver/bone/kid- ney（ALPL） gene--Plenti6.3-ALPL. METHODS： The effective sequence of ALPL gene was cloned into the pENTR - 2B vector using restriction endonuclease digestion and DNA ligation methods. Then the pENTR - 2B - ALPE entry vectors and the Plenti6.3/V5 - DEST lentiviral vectors were restructured. After DNA sequencing confirmation, the re- combinant lentiviral vectors were transfected into GP2 - 293T cells to pack lentivirus. The Plenti6.3 - ALPL virus was used to infect bone marrow mesenchymal stem cells （BMMSC） and the expression of ALPL on both RNA and protein levels was detected by ALP staining, RT- PCR and Western blot respectively. RESULTS： DNA sequencing and Western blot demonstrated that the lentivirus vector was constructed correctly. Moreover, ALP staining was enhanced in BMMSCs infected by the Plenti6.3-ALPL. CONCLUSION ： Recombinant lentiviral vector of ALPL gene was success- fully constructed.