hIFN-γ腺病毒表达载体的快速构建及鉴定

摘要

Objective To construct the IFN-γ adenvirus vector rapidly.Methods RNA of raji cell stimulated by IFN-γ(10 ng/ mL) was extracted using Trizol,and oligo primer was used for reverse transcription.The hIFN-γ cDNA sequence was acquired by PCR,inserted into vector pAdTrack-CMV,and the pAdTrack-CMV-hIFN-γ expression vector was obtained.Then the pAdEasy-1 was transferred into BJ5183 competent bacteria and screened by streptomycin sulfate and ampicillin resistance.pAdTrack-CMV-hIFN-γ was transferred into pAdEasy-1-BJ5183 competent bacteria and screened by kanamycin to obtain the pAdEasy-1-BJ5183-pAdTrack-CMV-hIFN-γ recombinant,and it was confirmed by Pac Ⅰ enzyme digestion.Results Successfully obtained the pAdEasy-1-pAdTrack-CMV-hIFN-γadenovirus expression vector.Conclusion adenorirus expression vecter construction efficiency by two-step medified methods,is much more efficient than traditional methods which transferred the linear pAdTrack-CMV-hIFN-γ and pAdEasy-1 into BJ5183 competent bacteria at one time,saving experimental time and materials.%目的 快速构建hIFN-γ腺病毒载体.方法 Trizol法提取IFN-γ(10 ng/mL)刺激的Raji细胞总RNA,以Oligo引物进行反转录获得cDNA序列,PCR法钓取人hIFN-γ的cDNA序列并插入pAdTrack-CMV载体中获得pAdTrack-CMV-hIFN-γ表达载体;然后将pAdEasy-1电转入BJ5183感受态菌并通过硫酸链霉素和氨苄青霉素双抗筛选获得pAdEasy-1-BJ5183感受态菌;之后将PmeI线性化的pAdTrack-CMV-hIFN-γ电转入pAdEasy-1-BJ5183感受态菌中并通过卡那霉素抗性筛选获得pAdEasy-1-B J5183-pAdTrack-CMV-hIFN-γ重组体;Pac Ⅰ酶切鉴定.结果 成功获得pAdEasy-1-pAdTrack-CMV-hIFN-γ腺病毒表达载体.结论 改良的两步法腺病毒表达载体的构建效率远高于同时将线性化pAdTrack-CMV-hIFN-γ与pAdEasy-1电转入BJ5183感受态菌以获得重组体传统方法,节省了实验时间和实验材料.