Ang2基因RNA干扰慢病毒表达载体的构建与鉴定

目的构建促血管生成素(Ang)2基因的RNAi慢病毒表达载体,并鉴定其正确性。方法将经XbaⅠ酶切电泳鉴定的pSilencer 1.0-U6-Ang2-siRNA重组质粒与经XbaⅠ酶切电泳鉴定的嗜中性多形核白细胞-绿色荧光蛋白转移质粒(pNL-EGFP)载体连接,产生pNL-EGFP-U6-Ang2-siR-NA慢病毒转移质粒,再以pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒、水疱性口炎病毒G蛋白包膜质粒和包装质粒三质粒共转染293T细胞,产生慢病毒,收集病毒上清液并测定病毒滴度。结果成功构建pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒2条,通过XbaⅠ酶切电泳及测序鉴定,证明Ang2-siRNA核苷酸序列插入的正确性。利用三质粒慢病毒包装系统生产EGFP-Ang2-siRNA病毒,收集病毒上清,并测得病毒滴度为9×103/μL。结论成功构建了Ang2基因的RNAi慢病毒表达载体,为下一步进行干扰体内外恶性黑素瘤Ang2的表达奠定基础。

Construction and Identification of RNAi Lentiviral Expression Vector for Ang2 Gene

Objective To construct and identify the RNAi lentiviral expression vector for angiopoietin（Ang）2 gene,and identify its validity.Methods pSilenser 1.0-U6-Ang2-siRNA reccombinant plasmid to ploymorphonuclear neutrophilic leukocyte-effective green fluorescence protein（pNL-EGFP）vector,which were digested and electorphoresis identified by using enzyme XbaⅠ,were joined to get pNL-EGFP-U6-Ang2-siRNA lentiviral transfer plamid,then the PNL-EGFP-U6-Ang2-siRNA lentiviral transfer plasmid,vesicular stomatitis virus G-protein envelope plasmid and pHelper packaging plasmid were cotransfected into 293T cells,resulting in lentivirus.The virus supernatant were collected and the viral titer was determined.Results The lentiviral transfer plasmids of pNL-EGFP-U6-Ang2-siRNA were constructed successfully and identified by using enzyme digestion electrophoresis of XbaⅠ and sequencing analysis,proved the correctness of the inserted Ang2-siRNA nudeotide sequence.EGFP-Ang2-siRNA virus were produced by using the three plasmid lentiviral packaging system.The virus supernatant was collected and viral titers measured for the 9×103/μL.Conclusion The RNAi lentiviral expression vectors for Ang2 gene were constructed successfully,and would be useful for the further research of the next research of interfering the Ang2 expression in malignant melanoma in vivo and vitro.