[1]孟春花,王春玲,李静心,等.猪APOBEC3F基因真核表达载体的构建及其在MARC145细胞中的表达定位[J].江苏农业学报,2015,(06):1344-1349.[doi:doi:10.3969/j.issn.1000-4440.2015.06.023]
　MENG Chun-hua,WANG Chun-ling,LI Jing-xin,et al.Construction of eukaryotic vector for porcine APOBEC3F gene and its expression and localization in MARC145 cells[J].,2015,(06):1344-1349.[doi:doi:10.3969/j.issn.1000-4440.2015.06.023]
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猪APOBEC3F基因真核表达载体的构建及其在MARC145细胞中的表达定位()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年06期
页码:
1344-1349
栏目:
畜牧兽医·水产养殖
出版日期:
2015-12-31

文章信息/Info

Title:
Construction of eukaryotic vector for porcine APOBEC3F gene and its expression and localization in MARC145 cells
作者:
孟春花12; 王春玲13; 李静心12; 李隐侠12; 朱前明13; 曹少先12
（1.江苏省农业科学院畜牧研究所，江苏南京210014；2.江苏省农业科学院动物品种改良与繁育重点实验室， 江苏南京210014;3.南京农业大学动物科技学院，江苏南京210095）
Author(s):
MENG Chun-hua12; WANG Chun-ling13; LI Jing-xin12; LI Yin-xia12; ZHU Qian-ming13; CAO Shao-xian12
(1.Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2.Key Laboratory of Animal Breeding and Reproduction, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;3.College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China)
关键词:
APOBEC3F; 真核表达载体; MARC145细胞; 转染; 荧光定量PCR; 脂质体; 猪
Keywords:
apolipoprotein B mRNA-editing enzyme cayalytic polypeptide 3F（APOBEC 3F）; eukaryotic expression vector; MARC145 cell; transfection; real time-PCR; liposome; swine〖WT〗〖KH+1*4D〗
分类号:
S858.65
DOI:
doi:10.3969/j.issn.1000-4440.2015.06.023
文献标志码:
A
摘要:
载脂蛋白B mRNA编辑酶催化多肽3F（APOBEC3F）属固有免疫系统中的重要成员，对人免疫缺陷病毒（HIV）、乙型肝炎病毒(HBV)等多种病毒的复制具有广泛的抑制作用。为构建APOBEC3F基因的真核表达载体，并检测其在体外培养的MARC145细胞中的表达情况，通过RT-PCR从猪脾脏组织扩增APOBEC3F基因，定向克隆到表达增强型绿色荧光蛋白（EGFP）的真核表达载体pEGFP-CMV中，构建pEGFP-APOBEC3F。PCR扩增及测序显示，APOBEC3F插入载体位置、方向及序列均正确。pEGFP-APOBEC3F经脂质体介导法转染MARC145细胞，转染后24 h APOBEC3F mRNA的水平升至最高。细胞免疫化学法检测发现APOBEC3F蛋白主要在MARC145细胞质中表达。研究结果为体外研究猪APOBEC3F基因在抗猪蓝耳病中的作用奠定了基础。
Abstract:
Apolipoprotein B mRNA-editing enzyme cayalytic polypeptide 3F (APOBEC3F，A3F) is an important member of innate immune system in mammals, playing vital roles in inhibiting the replication of multiple viruses, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), etc. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is one of the most economically important diseases of swine. No reliable or sustainable cure is available due to some special features of PRRSV. It is one of the effective directions to control PRRS disease by improving genetic resistance of the host. Our previous study has shown that the polymorphism of 〖WTBX〗A3F gene was associated with PRRSV resistance. In this study, 〖WTBX〗A3F gene was amplified by RT-PCR and cloned into pEGFP-CMV eukaryotic expression vector with enhanced green fluorescence protein (EGFP ) by using porcine splenic RNA as template and primers based on A3F gene sequences on GenBank. Verified by double enzymes (BglⅡ and SalⅠ) digestion and sequencing, A3F eukarytic expression vector was constructed and named pEGFP-〖WTBX〗A3F. The vector was then transfected into MARC145 cells mediated by liposome. Realtime PCR showed that the expression of 〖WTBX〗A3F mRNA was increased over time and reached its maximum 24 h after transfection and subcellular localization of A3F protein was in the cytoplasm revealed by immumocytochemistry (ICC). This research laid a foundation for the studies on A3F′s role in anti-PRRS in vitro.

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备注/Memo

备注/Memo:
收稿日期：2015-04-16 基金项目：江苏省自然科学基金项目（BK20140741）;江苏省农业科技自主创新基金项目[CX(12)2034]；国家自然科学基金项目（30972075） 作者简介：孟春花(1979-)，女，山东单县人，博士，副研究员。主要从事抗猪PRRSV育种的研究。(Tel)025-84390095;(E-mail)mengchunhua@jaas.ac.cn 通讯作者：曹少先，(Tel) 025-84390352;(E-mail)caoshaoxian@163.com
更新日期/Last Update: 2015-12-31