慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定

doi:10.3969/j.issn.1004-616x.2018.03.014

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (3): 234-238.doi: 10.3969/j.issn.1004-616x.2018.03.014

慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定

蔡艺煌¹, 邓小玲², 黄文霞¹, 黄洁², 纪晴¹, 许铭炎¹

1. 厦门医学院口腔系, 厦门医学院附属口腔医院, 福建厦门 361002;
2. 厦门大学医学院基础部, 福建厦门 361002

收稿日期:2018-02-03 修回日期:2018-03-07 出版日期:2018-05-30 发布日期:2018-05-30
通讯作者: 纪晴,E-mail:736681716@qq.com;许铭炎,E-mail:myxu@xmu.edu.cn E-mail:736681716@qq.com;myxu@xmu.edu.cn
作者简介:蔡艺煌,E-mail:caimorry@foxmail.com
基金资助:
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）

Construction and identification of lentiviral knockdown plasmid pLKO.1-hSRF

CAI Yihuang¹, DENG Xiaoling², HUANG Wenxia¹, HUANG Jie², JI Qing¹, XU Mingyan¹

1. Department of Stomatology, Affiliated Stomatological Hospital of Xiamen Medical College, Xiamen 361002;
2. Department of Basic Medical Science, Xiamen University Medical College, Xiamen 361002, Fujian, China

Received:2018-02-03 Revised:2018-03-07 Online:2018-05-30 Published:2018-05-30

摘要/Abstract

摘要： 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。

关键词: 慢病毒载体, 质粒构建, 血清反应因子, 口腔鳞癌细胞

Abstract: OBJECTIVE: To construct a plasmid which interfered with expression of the human SRF gene,and to study the role of SRF in oral squamous cell carcinoma. METHODS: A SRF gene-specific knockdown fragment was designed by using the Thermo Fisher's RNAi design tool,and then by double digestion of the pLKO.1 vector,gel extraction,and connection of specific fragment and the linearized vector by T4 ligase,then the pLKO.1-hSRF plasmid was obtained. The recombinant plasmids were identified by sequencing and restriction enzyme digestion. 293T cells were used to generate lentivirus by co-transfecting pLKO.1-hSRF with helper plasmids pVSV-G and pHR. The virus were collected and used to infect the oral squamous carcinoma cells,SAS. Stable cells were selected by puromycin and verified by Western blot and real-time quantitative PCR. RESULTS: The lentiviral knockdown plasmid pLKO.1-hSRF was identified by sequencing and restriction enzyme digestion. The expression of SRF protein and mRNA level of SAS cells infected with the lentiviral plasmid were significantly decreased compared with the control group. CONCLUSION: The lentiviral knockdown plasmid pLKO.1-hSRF was successfully constructed and the SAS cell line of low expression of SRF was obtained.

Key words: lentiviral vector, plasmid construction, serum response factor, oral squamous cell carcinoma

中图分类号:

R735.35

蔡艺煌, 邓小玲, 黄文霞, 黄洁, 纪晴, 许铭炎. 慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定[J]. 癌变·畸变·突变, 2018, 30(3): 234-238.

CAI Yihuang, DENG Xiaoling, HUANG Wenxia, HUANG Jie, JI Qing, XU Mingyan. Construction and identification of lentiviral knockdown plasmid pLKO.1-hSRF[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 234-238.

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慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定

Construction and identification of lentiviral knockdown plasmid pLKO.1-hSRF

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