猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用

doi:10.3969/j.issn.1004-1524.2021.02.05

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (2): 223-229.DOI: 10.3969/j.issn.1004-1524.2021.02.05

猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用

吴俊静(), 乔木, 周佳伟, 梅书棋, 彭先文^*()

动物胚胎工程及分子育种湖北省重点实验室,湖北省农业科学院畜牧兽医研究所,湖北武汉 430064

收稿日期:2020-05-20 出版日期:2021-02-25 发布日期:2021-02-25
作者简介:*彭先文,E-mail: pxwpal@163.com
吴俊静(1984—),女,湖北武汉人,博士,副研究员,主要从事猪遗传育种研究。E-mail: jeanne1106@126.com
通讯作者: 彭先文
基金资助:
湖北省重大科技创新项目(2019ABA084);湖北省重大科技创新项目(2017ABA134);湖北省创新群体项目(2018CFA014);湖北省农业科技创新中心创新团队项目(2019-620-000-001-18);国家生猪产业技术体系项目(CARS-36)

Function of porcine non-coding RNA lnc-000649 during PRRSV infection

WU Junjing(), QIAO Mu, ZHOU Jiawei, MEI Shuqi, PENG Xianwen^*()

Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Science, Wuhan 430064, China

Received:2020-05-20 Online:2021-02-25 Published:2021-02-25
Contact: PENG Xianwen

摘要/Abstract

摘要：

长链非编码RNA(lncRNA)在免疫及病毒与宿主互作中发挥重要作用。本研究在猪PAM细胞中发现一种新的猪长链非编码RNA lnc-000649,其全序列长1 483 bp。利用荧光定量PCR技术检测了猪繁殖与呼吸综合征病毒(PRRSV)感染前后PAM细胞中lnc-000649的表达水平,发现其在PRRSV感染后表达显著(P<0.01)下调。为了进一步研究该lncRNA的功能,构建了猪lnc-000649超表达载体lnc-000649-pcDNA3.1,并转染PAM细胞,成功构建了lnc-000649超表达细胞模型。经PRRSV接种后发现,转染lnc-000649-pcDNA3.1组的PAM细胞内PRRSV RNA含量极显著(P<0.01)低于转染pcDNA3.1(+)空载体和未转染组,说明猪lnc-000649对PRRSV增殖具有抑制作用,但是其具体作用机制还有待进一步研究。

关键词: 猪, 长链非编码RNA, 猪繁殖与呼吸综合征病毒, 超表达载体

Abstract:

Long noncoding RNA (lncRNA) plays a role in immunity and the host response to viral infection. In this study, a novel porcine lncRNA lnc-000649, with a total length of 1 483 bp, was found in porcine alveolar macrophage (PAM) cells. The expression level of lnc-000649 in PAM cells before and after PRRSV infection was detected by quantitative PCR, and it was found that the expression of lnc-000649 was significantly (P<0.01) down-regulated after PRRSV infection. In order to further study the function of this lncRNA, lnc-000649 overexpression vector lnc-000649-pcDNA3.1 was constructed and transfected into PAM cells. After PRRSV inoculation, the results showed that PRRSV RNA level in PAM cells which transfected with lnc-000649-pcdna3.1 was significantly (P<0.01) lower than that in transfected pcDNA3.1(+) and non-transfected group. It indicated that lnc-000649 could inhibit the proliferation of PRRSV. However, its specific mechanism remains to be further studied.

Key words: porcine, lncRNA, PRRSV, overexpression vector

中图分类号:

S828

吴俊静, 乔木, 周佳伟, 梅书棋, 彭先文. 猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用[J]. 浙江农业学报, 2021, 33(2): 223-229.

WU Junjing, QIAO Mu, ZHOU Jiawei, MEI Shuqi, PENG Xianwen. Function of porcine non-coding RNA lnc-000649 during PRRSV infection[J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 223-229.

图/表 7

表1 荧光定量PCR引物信息表

Table 1 Information of the qPCR primers

引物名称 Primer name	引物序列 Primer sequence (5'-3')	退火温度 Annealing temperature/℃	片段长度 Product length/bp
Lnc-000649 F	CGAGGAAGTGCTGATGTTG	55	184
Lnc-000649 R	TGGACCAGACGCTGAGAAT
β-actin F	CCAGGTCATCACCATCGG	55	158
β-actin R	CCGTGTTGGCGTAGAGGT
PRRSV F	CATTGCCAAACACCACTTTG	55	238
PRRSV R	CGCCAGTGTACATCACCATC

图1 PRRSV感染PAM细胞后猪lnc-000649的表达量变化 **表示差异极显著(P<0.01)。下同。

Fig.1 Expression levels of lnc-000649 in PRRSV-infected and mock control PAM cells ** represented the significant difference(P<0.01). The same as below.

图2 PAM细胞中猪lnc-000649的全长序列片段的PCR扩增图图中泳道M为DL2000 DNA marker;泳道1、2、3为猪lnc-000649的全长PCR扩增片段。

Fig.2 PCR amplification of the full-length fragment of porcine lnc-000649 in PAM cells Lane M was DL2000 DNA marker; Lane 1, 2, 3 were porcine lnc-000649 PCR product.

图3 猪lnc-000649超表达载体lnc-000649-pcDNA3.1双酶切鉴定结果泳道M为1 kb DNA ladder;泳道1为双酶切前的环形lnc-000649-pcDNA3.1载体;泳道2为lnc-000649-pcDNA3.1载体经HindⅢ和XbaⅠ双酶切后,线性化的pcDNA3.1(+)空载体片段与猪lnc-000649序列片段。

Fig.3 Identification result of porcine lnc-000649 overexpression vector lnc-000649-pcDNA3.1 by double enzyme digestion Lane M was 1 kb DNA ladder; Lane 1 was the annular Lnc-000649-pcDNA3.1 vector before enzyme digestion; Lane 2 was the linearized empty pcDNA3.1(+) vector and porcine lnc-000649 digested by HindⅢ and XbaⅠ.

图4 PAM细胞中猪lnc-000649荧光定量检测

Fig.4 Quantitative detection result of porcine lnc-000649 in PAMs

图5 PAM细胞中猪蓝耳病病毒(PRRSV)RNA定量检测结果

Fig.5 Quantitative detection result of PRRSV RNA in PAMs

图6 Marc-145细胞中猪蓝耳病病毒(PRRSV)滴度(TCID50)检测结果

Fig.6 PRRSV virus titer (TCID50) in Marc-145 cells

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猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用

Function of porcine non-coding RNA lnc-000649 during PRRSV infection

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