發(fā)布時間：2018-06-21 21:51

  本文選題：腺病毒 + 病毒核酸提取�。� 參考：《中國醫(yī)藥導(dǎo)報》2016年36期

【摘要】：目的建立一種簡單、快速、有效、純度高的人腺病毒全基因組提取的方法。方法用60 mm細(xì)胞培養(yǎng)皿培養(yǎng)A549或AD293細(xì)胞,接種培養(yǎng)人7型腺病毒(HAd V-7)CQ1198株,收集病毒感染的細(xì)胞,分別用Hirt's改良法、試劑盒A和試劑B提取HAd V-7-CQ1198病毒株全基因組,提取的基因組進行限制性內(nèi)切酶酶切實驗、細(xì)菌內(nèi)同源重組實驗和轉(zhuǎn)染細(xì)胞拯救病毒實驗。結(jié)果幾種方法都成功獲得高質(zhì)量的腺病毒全基因組,可以用于PCR、測序、酶切、同源重組和轉(zhuǎn)染實驗;兩種試劑盒方法較傳統(tǒng)的Hirt's改良法更快,不需要特別步驟去除細(xì)胞基因組,可以在1 h內(nèi)完成基因組提取。其中,試劑盒A提取的病毒基因組量最多(20~50μg),RNA含量最少,不需要另外加入核糖核酸酶(RNase),而Hirt's改良法需要在裂解液中或最終產(chǎn)物中加入RNase以去除細(xì)胞RNA成分。結(jié)論試劑盒A和試劑盒B均可替代傳統(tǒng)Hirt's提取方法用于快速提取高質(zhì)量腺病毒基因組,提取的基因組能用于酶切分型、轉(zhuǎn)染等實驗操作。
[Abstract]:Objective to establish a simple, rapid, effective and high purity genomic extraction method for human adenovirus. Methods A549 or AD293 cells were cultured in 60 mm cell culture dish. Human adenovirus type 7 (HAd V-7) CQ1198 strain was inoculated. The infected cells were collected. The whole genome of HAd V-7-CQ1198 strain was extracted by Hirtins modified method, kit A and reagent B, respectively. The extracted genomes were digested by restriction endonuclease, homologous recombination of bacteria and transfection of cells to save virus. Results High quality adenovirus genomes were successfully obtained by several methods, which could be used in PCR, sequencing, enzyme digestion, homologous recombination and transfection experiments. Genome extraction can be completed within 1 hour. The RNA content of the virus extracted from Kit A was the highest (20 ~ 50 渭 g), and RNase (RNase) was not needed. However, the improved method of HirtCs needed to add RNase to the lytic solution or the final product to remove the RNA components of the cells. Conclusion both Kit A and Kit B can be used for rapid extraction of high quality adenovirus genomes instead of the traditional HirttCys extraction methods. The extracted genomes can be used for enzyme digestion typing and transfection.
【作者單位】： 呼吸疾病國家重點實驗室廣州醫(yī)科大學(xué)附屬第一醫(yī)院;廣東省東莞市兒科研究所廣東省東莞市第八人民醫(yī)院;
【基金】：國家自然科學(xué)基金資助項目(31570163) 廣東省廣州市科技計劃項目(201504010032)
【分類號】：R373
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本文編號：2050135