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

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.

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