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慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定
Authors
纪晴
蔡艺煌
+4 more
许铭炎
邓小玲
黄文霞
黄洁
Publication date
31 May 2018
Publisher
Abstract
目的:构建敲减人血清反应因子SRF基因的慢病毒质粒,为进一步研究SRF在口腔鳞状细胞癌中的作用提供工具。方法:利用Thermo Fisher公司RNAi网站设计出针对SRF基因特异性敲减的片段,然后通过对pLKO.1载体进行双酶切,胶回收纯化后,经T4连接酶将双酶切线性化载体与设计的目的片段进行连接,转化和质粒提取,采用双酶切以及测序的方法对重组质粒进行序列鉴定。利用293T细胞对构建正确的pLKO.1-hSRF质粒进行病毒包装后感染SAS口腔鳞癌细胞,经嘌呤霉素筛选获得稳定株,并通过Western blot和实时荧光定量PCR对稳定转染细胞株的敲减效率进行验证。结果:构建出的慢病毒敲减质粒pLKO.1-hSRF经测序和酶切鉴定均正确,感染该慢病毒质粒的SAS细胞后,其SRF蛋白表达量和mRNA水平与对照组相比均显著下降(P<0.01)。结论:慢病毒敲减质粒pLKO.1-hSRF构建成功,获得SRF低表达的SAS细胞株。国家自然科学基金(81671001,81771079,207201);;福建省中青年骨干人才项目(2015-ZQN-ZD-35);;厦门医学院科研基金(K2015-06);;厦门市重大疾病联合攻关项目(3502Z20179051
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Last time updated on 10/06/2020