重组真核表达载体pcDNA3.1-Annexin A2的构建与鉴定

Construction of Eukaryon Expression Vector for pc DNA3. 1-Annexin A2

目的构建含Annexin A2目的基因的重组表达质粒。方法从鼻咽癌CNE-2细胞提取总RNA,RT-PCR扩增目的基因片段,经KpnⅠ和XbaⅠ双酶切后与pc DNA3.1(+)真核表达载体连接,构建真核表达载体pc DNA3.1-Annexin A2,转化DH5α,筛选阳性重组子,抽质粒进行KpnⅠand XbaⅠ双酶切、PCR和测序鉴定。结果重组质粒经PCR和酶切后都获得与RT-PCR大小相同的产物,长度约为1020b P,测序结果与基因库一致。结论成功构建重组真核表达质粒pc DNA3.1-Annexin A2。

OBJECTIVE To construct eukaryon expression vector for pcDNA 3.1-Annexin A2.METHODS Total RNA was extracted from nasopharyngeal carcinoma CNE-2 cells.The target gene Annexin A2 was amplified By RT-PCR.After KpnⅠand XbaⅠdouble digestion ,Annexin A2 was connected to eukaryotic Expression Vector pcDNA3.1（＋） by T4 DNA ligase and to construct the prokaryotic Expression plasmid pcDNA 3.1-Annexin A2,which was transformed into DH5α.The positive recombinants were identified by double digestion with restriction endonuclease KpnⅠ and XbaⅠ,PCR amplification and DNA sequencing.RESULTS A 1020bp fragment of the same size was obtained after recombinant plasmid pcDNA 3.1-Annexin A2 by PCR amplification and KpnⅠand XbaⅠdouble digestion.The sequencing results consisted with the genebank.CONCLUSION The eukaryotic expression plasmid for pcDNA3.1-Annexin A2 was successfully constructed.