稳定转染纤维素酶相关基因的猪胎儿成纤维细胞系的建立

畜牧兽医学报

稳定转染纤维素酶相关基因的猪胎儿成纤维细胞系的建立

黄妙容^1，3，张献伟¹，刘德武¹，邹娴¹，帅亮¹，袁玉娟¹，朱财林¹，吴珍芳^1,2*

1.华南农业大学动物科学学院，广州 510642； 2.广东省温氏集团研究院，新兴 527400；3. 肇庆大华农生物药品有限公司，广东省兽用生物制品技术研究与应用企业重点实验室，肇庆 526238

收稿日期:2012-07-30 出版日期:2013-04-23 发布日期:2013-04-23
通讯作者: 吴珍芳，教授，E-mail: wzfemail@163.com
作者简介:黄妙容(1983-)，女，广东潮州人，博士，主要从事动物克隆与转基因育种的研究，Tel：020-85284833，E-mail: miaorongh@yahoo.com.cn
基金资助:
国家高技术研究发展计划(863)项目(2011AA100304)；广东省自然科学基金项目(2011A020102003)；国家科技重大专项(2011ZX08006004)；粤港关键领域重点突破项目(2008A024200012)

Establishment of Transgenic Porcine Fetal Fibroblast Cell Line Stably Transfected with Fibrolytic Enzymes Synthesis Genes

HUANG Miao-rong^{1, 3}, ZHANG Xian-wei¹, LIU De-wu¹, ZOU Xian¹, SHUAI Liang¹, YUAN Yu-juan¹, ZHU Cai-lin¹, WU Zhen-fang^{1, 2*}

1. College of Animal Science, South China Agricultural University, Guangzhou 510642, China;2. Institute of Wen's Group, Guangdong Province, Xinxing 527400, China; 3. Key Laboratory of Biotechnology R&D of Veterinary Biology Products of Guangdong Enterprise, Zhaoqing Dahuanong Biology Medicine Co., Ltd，Zhaoqing 526238, China

Received:2012-07-30 Online:2013-04-23 Published:2013-04-23

摘要/Abstract

摘要：

旨在构建带有双筛选标记的纤维素酶基因的腮腺特异性表达载体，筛选稳定转染该载体的猪胎儿成纤维细胞系，为制备转纤维素酶基因的转基因克隆猪提供核供体细胞。采用SOE-PCR和PCR方法，分别扩增获得筛选标记基因NEO-T2A-EGFP序列和纤维素酶相关基因的融合序列SP-EGX-F2A-BGL1，构建腮腺特异性表达载体pPSP-SP-EGX-F2A-BGL1-CMV-NEO-T2A-EGFP，经PCR、酶切、测序鉴定正确后，通过脂质体转染猪胎儿成纤维细胞，利用G418抗性和绿色荧光蛋白进行稳定筛选，并利用PCR和Western blot进行检测。结果表明，成功构建了带双筛选标记的腮腺特异表达纤维素酶基因的表达载体，并获得稳定转染该载体的猪胎儿成纤维细胞系。该结果为生产腮腺特异表达纤维素酶的转基因克隆猪研究奠定基础。

Abstract:

The aim of the present study was to construct a salivary-glands-specific expression vector, which including dual screen markers gene and fibrolytic enzymes gene, and then it was transfected into porcine fetal fibroblast cells to provide fibrolytic enzymes transgenic donor cells for cloned pigs. The sequences of dual screen marker gene NEO-T2A-EGFP and fibrolytic enzyme gene SP-EGX-F2A-BGL1 were amplified by SOEPCR and PCR, respectively, and inserted into the salivary-glands-specific expression vector to generate pPSP-SP-EGX-F2A-BGL1-CMV-NEO-T2A-EGFP. After identification by PCR, restrictive enzymes digestion and sequencing, the vector was transfected into porcine fetal fibroblast cells with liposome, and the stably transfected cells were screened by G418 selection and EGFP and identified by PCR and Western blot analysis. The result showed that the salivary-glands-specific expression vector with dual screen marker genes and fibrolytic enzyme genes was constructed, and the stable transgenic porcine fetal fibroblast cell lines were established. The study paves the way for further research on production of cloned pig expressing fibrolytic enzymes in their salivary glands.

中图分类号:

S828
S814.1

黄妙容，张献伟，刘德武，邹娴，帅亮，袁玉娟，朱财林，吴珍芳. 稳定转染纤维素酶相关基因的猪胎儿成纤维细胞系的建立[J]. 畜牧兽医学报, doi: .

HUANG Miao-rong, ZHANG Xian-wei, LIU De-wu, ZOU Xian, SHUAI Liang, YUAN Yu-juan, ZHU Cai-lin, WU Zhen-fang. Establishment of Transgenic Porcine Fetal Fibroblast Cell Line Stably Transfected with Fibrolytic Enzymes Synthesis Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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