ERGIC3的稳定表达细胞株的建立与鉴定

ERGIC3的稳定表达细胞株的建立与鉴定

郑翔，刘兴宇，李学英，吴明松*

（遵义医学院细胞生物学与遗传学教研室，贵州遵义563000）

[摘要] 目的构建ERGIC3基因的真核表达载体，通过将载体转染支气管上皮细胞株BEAS-2B，筛选并建立ERGIC3稳定表达的细胞株，为进一步研究ERGIC3在肺癌中的作用奠定基础。方法采用RT-PCR方法获取人类ERGIC3基因的ORF 框的DNA序列；构建逆转录病毒载体pLXSN-ERGIC3，用PT-67细胞包装后获得病毒颗粒；用病毒颗粒感染BEAS-2B细胞；通过G418筛选获取整合了外源基因ERGIC3的细胞；单克隆化成为稳定转染ERGIC3细胞株；然后用定量RT-PCR和Western blot进行鉴定后，用划痕实验研究稳定转染细胞株的迁移能力和形态学变化。结果获得了4个ERGIC3稳定表达细胞株，这些细胞株的ERGIC3表达量远高于对照组细胞。稳定转染细胞株的迁移能力显著增强（P<0.05）。结论成功的构建了ERGIC3基因的真核表达载体pLXSN-ERGIC3；筛选出ERGIC3基因稳定转染支气管上皮细胞株且其迁移能力明显增加。

[关键词] ERGIC3；稳定转染；真核表达；肺癌

[中图分类号] Q782[文献标志码] A Construction and identification of cell lines with ERGIC3 gene stable transfection

ZHENG Xiang, LIU Xing-yu, LI Xue-ying, WU Ming-song

(Department of Cell Biology and Genetics, Zunyi Medical University, Guizhou Zunyi 563000, China)

[Abstract]Objective To construct a eukaryotic expression vector carrying human gene ERGIC3 and to obtain human bronchial epithelial cell line stably transfected with ERGIC3, which provides the foundation of ERGIC3 in lung cancer. Methods The pLXSN retroviral vector was inserted the DNA fragment of open reading frame sequence (ORF) of gene ERGIC3 which obtained by PCR method. After the constructed pLXSN vector was transfected into the packaging cell, PT67 cell line for 48 hours, the replication-incompetent retroviral particles were harvested and infected the BEAS-2B cells. Screened with G418，the single cell clones were sought out and cultured which were stable transfection cell lines. Then the migration of the cell lines was tested by wound healing scratch assay. Results The vector carrying ERGIC3 was successfully constructed and 4 stable transfection cell lines were obtained, as well as the ERGIC3 expressions were higher than the control by real-time PCR and Western blot. The ability of the stable transfected cell line with ERGIC3 was significantly faster than the control cell (P<0.05). Conclusion The stable cell lines with expression [基金项目] 贵州省科学技术基金（黔科合J字[2013]2319号）；遵义医学院博士启动基金（F-655）。

[通讯作者] 吴明松，男，博士，研究方向：肿瘤细胞分子生物学，E-mail：

hanyue4187@。

ERGIC3 have been successfully constructed and the migration ability of the cells increased significantly.

[Key words] ERGIC3；Stable transfection；Eukaryotic expression；Lung cancer ERGIC3主要参与蛋白质从内质网到高尔基体的早期运输，近年的研究显示，ERGIC3在肺癌、肝癌中异常高表达，还参与肺癌[1]、肝癌[2]的发生发展，以及抑制BFA诱导的HEK-293细胞凋亡[3]。然而机制尚不清楚。大多数肺癌起源于支气管上皮细胞[4]，因此我们构建了pLXSN-ERGIC3真核表达载体，并将其转染入支气管上皮细胞，筛选出稳定转染ERGIC3的支气管上皮细胞株，为进一步研究ERGIC3基因在肺癌中的作用奠定实验基础。

1材料与方法

1.1 试验材料

BEAS-2B细胞、PT-67细胞由中科院昆明动物所曹毅研究员惠赠。pLXSN 载体购自美国Clontech公司，DMEM培养基购自Gibco公司，胎牛血清购自杭州四季青生物有限公司，SYB Green购自Invitrogen公司，逆转录酶购自Promega 公司，Taq酶购自Sigma公司，定量PCR仪购自Bio-rad公司。

1.2 细胞培养

BEAS-2B细胞和PT-67细胞生长于含10% 胎牛血清DMEM培养基中，置于37℃、5% CO2的培养箱中培养。2～3天换液，3～5天传代，细胞处于对数生长期时用于实验。

1.3 引物设计及合成

参照GenBank的人ERGIC3基因序列（NM_198398.1），用Primer 5.0 软件设计PCR引物，P1：5'-GGTGGTGAATTCATGAGGCGCTGGGGAAGCT-3'，P2：5'-GGTGGTGGATCCACCGAGGAGGGTGACTACGTTGTCTT-3'，在引物的5'端分别加上保护碱基GGTGGT和EcoR I、BamH I的酶切序列。定量PCR引物：ERGIC3: F: 5'-GGAGAGGTACTGAGGACAAATCA-3'，R: 5'-AGCTCATAGAGGACGAAGACTC-3'; Actin: F: 5'-CGGGAAATCGTGCGTGAC-3'，R: 5'-CAGGA AGGAAGGCTGGAAG-3'。引物由上海生物工程有限公司合成。

1.4 人ERGIC3基因的扩增

用TRIzol试剂提取肺组织总RNA，以其为模板，随机引物为引物，用逆转