鸡MMP7稳定过表达DF-1细胞系的构建与鉴定

doi:10.11843/j.issn.0366-6964.2025.12.045

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6442-6449.doi: 10.11843/j.issn.0366-6964.2025.12.045

鸡MMP7稳定过表达DF-1细胞系的构建与鉴定

邓梦玲¹(), 隋敏敏¹^,², 宋海洋³, 杨建发⁴, 邹丰才¹^,⁴^,*(), 贺君君⁴^,*()

1. 云南农业大学动物科学技术学院, 昆明 650201
2. 云南农业职业技术学院畜牧兽医学院, 昆明 650201
3. 温州科技职业学院动物科学学院, 温州 325000
4. 云南农业大学动物医学院, 昆明 650201

收稿日期:2025-03-27 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 邹丰才,贺君君 E-mail:merlideng@163.com;zfc1207@vip.163.com;hejunjun617@163.com
作者简介:邓梦玲(1989-)，四川德阳人，博士生，主要从事动物寄生虫及其致病性研究，E-mail：merlideng@163.com
基金资助:
NSFC-云南省联合基金项目(U2202201)；云南省家畜疫病病原生物学重点实验室(202449ce340019)；云南省教育厅科学研究基金项目(2025j1508)；云南省国际科技特派员项目(202403ak140036)

Construction and Identification of a Chicken MMP7 Stable Overexpression DF-1 Cell Line

DENG Mengling¹(), SUI Minmin¹^,², SONG Haiyang³, YANG Jianfa⁴, ZOU Fengcai¹^,⁴^,*(), HE Junjun⁴^,*()

1. Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
2. Institute of Animal Husbandry, Yunnan Vocational College of Agriculture, Kunming 650201, China
3. School of Animal Science, Wenzhou Vocational College of Science and Technology, Wenzhou 325000, China
4. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Received:2025-03-27 Online:2025-12-23 Published:2025-12-24
Contact: ZOU Fengcai, HE Junjun E-mail:merlideng@163.com;zfc1207@vip.163.com;hejunjun617@163.com

摘要/Abstract

摘要：

旨在构建稳定过表达鸡mmp7基因的DF-1细胞系，为细胞水平研究mmp7基因在相关疾病中的功能奠定基础。合成鸡mmp7基因序列，构建重组慢病毒质粒pLVX-MMP7-IRES-puro，利用三质粒慢病毒包装系统，将pLVX-MMP7-puro与辅助质粒pCMV-VSV-G、psPAX2共转染至对数生长期的293T细胞，获得携带mmp7基因序列的重组慢病毒，重组慢病毒感染DF-1细胞72 h后, 使用含1.5 μg·mL^-1嘌呤霉素的筛选，通过PCR验证mmp7基因在DF-1细胞基因组中的整合情况，RT-PCR检测mmp7基因的转录水平，以及Western blot分析MMP7蛋白的表达水平，以鉴定是否成功获得稳定过表达鸡MMP7的DF-1细胞系，并使用CCK-8试验评估过表达MMP7对DF-1细胞增殖的影响。本研究成功构建了稳定过表达鸡MMP7的DF-1细胞系，过表达MMP7在24~48 h对细胞活力无明显影响，但120~144 h后细胞活力显著降低(P < 0.05)。该细胞系的构建为后续深入探究MMP7的生物学功能提供了重要工具。

关键词: 基质金属蛋白酶7, 慢病毒, 稳定过表达, DF-1细胞系

Abstract:

This study aimed to establish a DF-1 cell line stably overexpressing chicken mmp7 gene, providing a foundation for investigating the role of mmp7 in related diseases at the cellular level. The chicken mmp7 gene sequence was synthesized and cloned into the lentiviral vector pLVX-MMP7-IRES-puro. Using a three-plasmid lentiviral packaging system, the recombinant plasmid pLVX-MMP7-puro was co-transfected with the helper plasmids pCMV-VSV-G and psPAX2 into 293T cells at the logarithmic growth phase to produce mmp7-carrying lentiviral particles. DF-1 cells were infected with the recombinant lentivirus for 72 h, followed by puromycin selection (1.5 μg·mL^-1). Integration of mmp7 into the DF-1 genome was confirmed by PCR, while its transcriptional and protein expression levels were assessed via RT-PCR and Western blot, respectively. The CCK-8 assay was used to evaluate the effect of MMP7 overexpression on DF-1 cell proliferation. A DF-1 cell line stably overexpressing chicken MMP7 was successfully generated. Overexpression of MMP7 showed no significant impact on cell viability at 24-48 h but led to a notable reduction at 120-144 h (P < 0.05). This cell line serves as a valuable tool for further exploration of MMP7's biological functions.

Key words: matrix metalloproteinase-7, lentivirus, stable overexpression, DF-1 cell line

中图分类号:

邓梦玲, 隋敏敏, 宋海洋, 杨建发, 邹丰才, 贺君君. 鸡MMP7稳定过表达DF-1细胞系的构建与鉴定[J]. 畜牧兽医学报, 2025, 56(12): 6442-6449.

DENG Mengling, SUI Minmin, SONG Haiyang, YANG Jianfa, ZOU Fengcai, HE Junjun. Construction and Identification of a Chicken MMP7 Stable Overexpression DF-1 Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6442-6449.

图/表 4

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鸡MMP7稳定过表达DF-1细胞系的构建与鉴定

Construction and Identification of a Chicken MMP7 Stable Overexpression DF-1 Cell Line

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