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目的 构建单羧酸转运泵基因反义逆转录病毒真核表达载体。方法 应用RT PCR方法扩增带有双酶切位点的单羧酸转运泵目的基因片段 ,与带有相同酶切位点的pLXSN逆转录病毒真核表达载体粘末端进行反向基因插入连接 ,构建具有反义表达的逆转录病毒载体。结果 构建载体进行双酶切电泳分析及插入片段基因序列分析 ,证明反义载体构建成功。结论 应用RT PCR方法扩增插入DNA片段 ,简单、灵活、快捷。双酶切定向连接构建反义表达载体可实现构建一步到位 ,免去正反向筛选的麻烦。
Objective To construct a monocarboxylate transporter gene antisense retrovirus eukaryotic expression vector. Methods The target gene fragment of monocarboxylate transporter with double restriction enzyme sites was amplified by reverse transcription polymerase chain reaction (RT PCR) and inserted into the end of the retroviral vector of pLXSN retrovirus eukaryotic expression vector. Construction of retroviral vector with antisense expression. Results The constructed vector was analyzed by double enzyme digestion electrophoresis and sequence analysis of inserted fragment gene to prove that the antisense vector was successfully constructed. Conclusion The PCR amplification of inserted DNA fragments is simple, flexible and rapid. Double restriction endonuclease construction of antisense expression vector can be constructed in one step, eliminating the trouble of forward and reverse selection.