人BMP2-IRES-HIF1αmu腺病毒表达载体的构建及其在HEK293细胞中的表达

摘要

目的 构建人BMP2-IRES-HIF1 αmu腺病毒表达载体,并转染HEK293细胞,为下一步转染骨髓基质细胞和体内实验打下基础.方法 PCR扩增HIF1αmu片段,用BstXⅠ和XbaⅠ双酶切回收目的片段.pIRES2-EGFP用BstXⅠ和Xba Ⅰ进行双酶切后回收大片段.将上述回收的目的基因与载体片段连接,然后转化感受态大肠杆菌DH5α扩增；PCR扩增BMP2片段,用Nhe Ⅰ和BamH Ⅰ双酶切后回收目的片段.把目的基因与载体片段连接,转化感受态大肠杆菌DH5α扩增重组腺病毒表达载体,通过酶切分析、PCR和测序进行鉴定.将构建好的质粒转染HEK293细胞,检测病毒液滴度.结果 构建了人BMP2-1RES-HIF1 αmu腺病毒表达载体,转染HEK293细胞见绿色荧光表达.结论 成功构建了人BMP2 -IRES-HIF1 αmu腺病毒表达载体,酶切分析及DNA测序证实质粒构建正确,质粒成功转染HEK293细胞,并见绿色荧光蛋白表达.%Objective To construct and identify human BMP2-IRES-HIFlotmu Adenovirus expressing carrier, trans-feet it in HEK293 cells, and determinate the virus droplet degrees. Methods PCR was used to amplify HIFlamu segments, BstX I and Xba I double enzyme cut pIRES2-EGFP and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification. PCR was used to amplify BMP2 segments, Nhe I and BamH I double enzyme cut and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification and restructuring adenovirus expressing carrier. Using the enzyme cut analysis, PCR for identification. The correct recombinant express plasmid was transfected into HEK293 cells, and detecting the virus droplet degrees. Results The adenovirus shuttle plasmid was constructed. Green fluorescent expression was seen in transfected HEK293 cells. Conclusion The adenovirus shuttle plasmid is constructed, it is successfully expressed in HEK293 cells and GFP expression is found.