SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达

王江; 段君君; 张小玉; 贾霄; 汪小波; 何颖红

SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达

王江^1,2,
段君君^1,2,
张小玉^1,2,
贾霄^1,2,
汪小波^1,2,
何颖红^1,2

1. 大理大学基础医学院
2. 云南省高校细胞生物学重点实验室

基金项目:

基金：国家自然科学基金资助项目 (31360288);

计量
- 文章访问数: 1558
- HTML全文浏览量: 537
- PDF下载量: 84
- 被引次数: 0
出版历程
- 收稿日期: 2017-10-23

Construction of Lentiviral Expression Vector Carrying SOX9 Gene and Its Stable Expression in LNcap Cell

Wang Jiang ^1,2,
Duan Jun Jun ^1,2,
Zhang Xiao Yu ^1,2,
Jia Xiao ^1,2,
Wang Xiao Bo ^1,2,
He Ying Hong ^1,2

Funds:

基金：国家自然科学基金资助项目 (31360288);

摘要

摘要: 目的构建SOX9基因慢病毒表达载体, 建立稳定表达SOX9的LNCa P细胞株.方法 PCR扩增SOX9基因, 将其克隆到慢病毒表达载体p LVX-IRES-Puro中, 双酶切和测序鉴定重组质粒.用HEK293细胞包装重组病毒颗粒并感染LNcap细胞, 经puromycin筛选获得稳定转染细胞株.q RT-PCR和Western Blot分别检测SOX9 m RNA水平及蛋白表达.结果经双酶切和测序鉴定证实目的片段已插入到慢病毒表达载体上.重组质粒转入LNCa P细胞后经puromycin抗性筛获得稳定表达的细胞株, q RT-PCR检测到SOX9基因在转录水平的表达, Western blot分析显示SOX9蛋白高表达.结论慢病毒表达载体p LVX-IRES-FLAG-SOX9构建成功, 实现了SOX9在LNcap细胞中的外源性表达.
- SOX9 /
- 慢病毒表达载体 /
- LNcap细胞 /
- 脂质体转染
Abstract: Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9. Me thods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector p LVX-IRES-Puro. p LVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening.The m RNA level and protein level in the infected LNcap cell were detected by q RT-PCR and Western blot respectively. Re s ults Restriction enzyme digestion and sequencing demonstrated that SOX9 c DNA was successfully cloned into p LVX-IRES-FLAG lentiviral vector. After the transfection to LNCa P cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 m RNA detected by q RT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclus ion The recombinant lentiviral vector bearing human SOX9 c DNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.
- SOX9 /
- Lentiviral vector /
- LNcap cell /
- Liposome tansfection

HTML全文

参考文献(15)

[1]	[1] BERTA P, HAWKINS J R, SINCLAIR A H, et al.Genetic evidence equating SRY and the testis-determining factor[J].Nature, 1990, 348 (6300) :448-450.
[2]	[2]AKIYAMA H.Control of chondrogenesis by the transcription factor Sox9[J].Mod Rheumatol, 2008, 18 (3) :213-219
[3]	[3]WANG X, JU Y, ZHOU M I, et al.Upregulation of SOX9promotes cell proliferation, migration and invasion in lung adenocarcinoma[J].Oncol Lett, 2015, 10 (2) :990-994.
[4]刘庆珍, 吴尽, 邳晓盟, 等.软骨发育过程中负调控SOX9基因表达机制研究进展[J].农业科学与技术 (英文版) , 2017, 18 (6) :1038-1041.
[5]	[5] SUN W, ZHANG K, LIU G, et al.SOX9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model[J].PLo S One, 2014, 9 (4) :e93570.
[6]	[6]WUERTZ K, GODBURN K, IATRIDIS J C.MSC response to p H levels found in degenerating intervertebral discs[J].Biochemical and Biophysical Research Communications, 2009, 379 (4) :824-829.
[7]王洪月, 李婷, 曾峥, 等.转录因子SOX9与肿瘤发生的关联[J].国际遗传学杂志, 2017, 40 (4) :221-227.
[8]	[8]WILHELM D, MARTINSON F, BRADFORD S, et al.Sertoli cell differentiation is induced both cell-autonomously and through prostaglandin signaling during mammalian sex determination[J].Developmental biology[J].2005, 287 (1) :111-124.
[9]	[9] WAGNER T, WIRTH J, MEYER J, et al.Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9[J].Cell, 1994, 79 (6) :1111-1120.
[10]	[10] VETRO A, CICCONE R, GIORDA R, et al.XX males SRY negative:a confirmed cause of infertility[J].J Med Genet, 2011, 48 (10) :710-712.
[11]	[11] ZHU D, CHEN H, YANG X, et al.mi R-32 functions as tumor suppressor and directly targets SOX9 in human non-small cell lung cancer[J].Onco Targets Ther, 2015, (8) :1771-1783.
[12]	[12] FAZILATY H, GARDANEH M, AKBARI P, et al.SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer[J].Cancer Microenviron, 2016, 9 (1) :71-74.
[13]	[13]LARSIMONT J C, YOUSSEF K K, SANCHEZ-DANES A, et al.SOX9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion[J].Cell Stem Cell, 2015, 17 (1) :60-73.
[14]汤晗, 聂建云, 李文斌, 等.慢病毒载体系统的发展及在肿瘤基因治疗中的应用[J].现代肿瘤医学, 2017, 25 (10) :1659-1662.
[15]	[15] HAN G Z, WOROBEY M.Endogenous lentiviral elements in the weasel family (Mustelidae) [J].Mol Biol Evol, 2012, 29 (10) :2905-2908.

相关文章(20)

[1]	刘晓琳, 谢青昕, 李漂, 张茜, 杨艾, 鲁卫东. 流感疫苗脂质体的制备方法及粒径对免疫原性的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220508
[2]	李漂, 刘晓琳, 杨艾, 张茜, 鲁卫东. PEG6000修饰的流感疫苗脂质体的制备和稳定性, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220307
[3]	毛瑞, 何影, 代自超, 李松. SOX9在SD大鼠胚胎发育髁突软骨与胫骨生长板软骨中的时间表达研究, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220210
[4]	刘艳红, 赵祥月, 谢青昕, 普梦笛, 鲁卫东. DC-Chol阳离子脂质体佐剂对流感疫苗免疫效果的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210302
[5]	范宇超, 付围, 普梦笛, 赵祥月, 鲁卫东. 不同稀释浓度对狂犬疫苗脂质体免疫原性的影响, 昆明医科大学学报.
[6]	王福科, 张红, 杨桂然, 肖渝, 何川, 宋恩, 李彦林. 携载增强型绿色荧光蛋白基因慢病毒转染骨髓间充质干细胞示踪方法, 昆明医科大学学报.
[7]	罗云, 罗钰辉, 刘孝东, 崔庆鹏. 大鼠CaSR基因的慢病毒高表达细胞系和RNA干扰质粒的构建及鉴定, 昆明医科大学学报.
[8]	兰萍, 叶红艳, 付围, 范宇超, 赵祥月, 鲁卫东. 狂犬疫苗脂质体稳定性及稀释比例对脂质体干粉的免疫原性, 昆明医科大学学报.
[9]	段君君, 方峰, 张小玉, 王江, 贾霄, 何颖红. WDR5真核表达载体的构建及其在LNCaP细胞中的稳定表达, 昆明医科大学学报.
[10]	戈佳云. 慢病毒介导的FHIT基因过表达调控人肝癌细胞株生长实验研究, 昆明医科大学学报.
[11]	杨洁. 转录因子Slug和Sox9对鼻咽癌干细胞侵袭迁移能力的实验研究, 昆明医科大学学报.
[12]	乔建斌. 流感疫苗壳聚糖脂质体的制备工艺, 昆明医科大学学报.
[13]	郑志. Syncytin慢病毒干扰载体及syncytin沉默Jurkat细胞系的建立, 昆明医科大学学报.
[14]	杜明. MMP-9表达在不同方法获得破骨细胞的比较研究, 昆明医科大学学报.
[15]	谷万港. HIV-1 整合酶真核表达载体的构建及在Hela细胞中的表达和定位, 昆明医科大学学报.
[16]	陈绍春. 用慢病毒转染技术实现Gnaq基因在SH-SY5Y细胞中持续稳定高表, 昆明医科大学学报.
[17]	刘为军. 慢病毒载体介导CD1d和GFP融合基因转染PANC-1细胞的实验研究, 昆明医科大学学报.
[18]	. 人乳头状瘤病毒16型E6基因短发夹结构RNA慢病毒载体的构建及鉴定, 昆明医科大学学报.
[19]	梁平. 人参皂苷Rg1脂质体的制备及稳定性研究, 昆明医科大学学报.
[20]	朱兴宝. ORMA40载体介导生存素基因转染嗅神经鞘细胞, 昆明医科大学学报.