鸡marco稳转巨噬细胞系的构建与鉴定

doi:10.11843/j.issn.0366-6964.2024.11.046

摘要/Abstract

摘要：

旨在通过慢病毒表达系统构建稳定表达marco基因的HD11细胞系，为研究marco基因的抗病毒免疫功能提供重要工具。首先，以HD11细胞为模板，通过PCR扩增获得marco基因编码区序列，并将其克隆到慢病毒载体上，获得重组慢病毒质粒pLV3-CMV-MCS-Puro-marco；其次，利用四质粒慢病毒包装系统将重组质粒pLV3-CMV-MCS-Puro-marco与辅助质粒pLP1、pLP2、pMD2.G共转染至293T细胞进行慢病毒包装，构建含鸡marco基因的重组慢病毒。最后，用包装成功的重组慢病毒感染HD11细胞72 h后进行嘌呤霉素(2 μg·mL^-1)筛选，通过RT-qPCR检测marco基因表达后最终确认获得稳定表达鸡marco基因的HD11细胞系。本研究成功构建了稳定表达鸡marco基因的HD11细胞系，为后续深入研究marco基因的功能提供了重要基础。

关键词: marco细胞系, 稳定表达, 慢病毒

Abstract:

The study aimed to establish the HD11 cell line with a stable expression of the marco gene through the lentivirus expression system, providing a crucial tool for investigating the antiviral immune function of the marco gene. Initially, the marco gene coding sequence was amplified by PCR using HD11 cells as a template and cloned into the lentiviral vector to generate the recombinant lentiviral plasmid pLV3-CMV-MCS-Puro-marco. Subsequently, the recombinant plasmid pLV3-CMV-MCS-Puro-marco was co-transfected with helper plasmids pLP1, pLP2, and pMD2.G into 293T cells using a tetra-plasmid lentivirus packaging system, resulting in the construction of recombinant lentivirus carrying the chicken marco gene. The successfully packaged recombinant lentivirus was then used to infect HD11 cells, followed by puromycin (2 μg·mL^-1) selection for 72 h. The stable expression of the chicken marco gene in the HD11 cell line was confirmed by RT-qPCR analysis. This study successfully established a stable HD11 cell line expressing the chicken marco gene, providing a crucial basis for further exploration of the marco gene's function.

Key words: marco cell line, stable expression, lentivirus

中图分类号:

S831.2

郭旺, 彭光众, 黄宏奥, 吴慧显, 胡序明, 张钰, 张扬, 陈国宏, 徐琪. 鸡marco稳转巨噬细胞系的构建与鉴定[J]. 畜牧兽医学报, 2024, 55(11): 5310-5316.

Wang GUO, Guangzhong PENG, Hongao HUANG, Huixian WU, Xuming HU, Yu ZHANG, Yang ZHANG, Guohong CHEN, Qi XU. Construction and Identification of the Chicken marco-stable Macrophage Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5310-5316.

图/表 4

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