WO2020024313A1 - Marco在筛选抗蓝耳病猪中的应用 - Google Patents

Marco在筛选抗蓝耳病猪中的应用 Download PDF

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WO2020024313A1
WO2020024313A1 PCT/CN2018/099287 CN2018099287W WO2020024313A1 WO 2020024313 A1 WO2020024313 A1 WO 2020024313A1 CN 2018099287 W CN2018099287 W CN 2018099287W WO 2020024313 A1 WO2020024313 A1 WO 2020024313A1
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marco
prrsv
pigs
resistant
blue ear
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French (fr)
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郭春和
张晓晓
朱振邦
俞飘
董文娟
贺胜
王小瑛
刘小红
陈瑶生
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中山大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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  • the invention belongs to the technical field of biomedicine. More specifically, it relates to the use of MARCO in screening for pigs resistant to blue ear disease.
  • Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease, is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
  • PRRS Porcine Reproductive and Respiratory Syndrome Virus
  • OIE World Organisation for Animal Health
  • PRRS is one of the most important infectious diseases in the pig industry. It is distributed in almost all pig countries and causes huge economic losses to the pig industry worldwide.
  • porcine high fever (PHFS) broke out in most provinces in China and Vietnam, causing huge economic losses to the pig industry.
  • the disease was caused by mutant PRRSV and was named highly pathogenic pigs.
  • Reproductive and respiratory syndrome virus Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).
  • HP-PRRSV is characterized by high fever, high pathogenicity, and high mortality.
  • the Ministry of Agriculture has included the disease in the mandatory immunization plan for major animal diseases.
  • PRRSV prevention and control is a difficult problem in China and the world.
  • the difficulty in preventing and controlling PRRSV is mainly manifested in the following aspects: (1) Macrophages and immunosuppressive diseases.
  • PRRSV mainly infects porcine alveolar macrophages (PAMs).
  • PAMs are immune cells and destroy PAMs. As a result, the body's immune system is destroyed, which leads to immunosuppression;
  • PRRSV vaccines on the market have almost no cross-protection power, and there is no cross-protection power between different strains; (4) enhanced antibody dependency, PRRSV Infection will stimulate the body to produce antibodies, but high titer antibodies will not only neutralize the virus, but will promote the proliferation of the virus; (5) persistent virus infection, PRR After SV infection, viremia can be detected in pigs for a long time, and PRRSV lasts for up to 5 months in the body; (6) Mixed infections. At present, mixed infections of blue ears and other diseases are more common in clinical practice, especially circular Viruses, Haemophilus parasuis, swine pneumonia and other mixed infections with PRRSV make it difficult to prevent and control PRRSV.
  • the technical problem to be solved by the present invention is to overcome the deficiencies and deficiencies of the existing pig blue ear disease prevention technology, and provide a gene related to pig blue ear disease resistance.
  • the research of the present invention shows that PRRSV inhibits the expression of MARCO after infection of PAMs cells, Overexpression inhibits PRRSV replication, and MARCO interference promotes PRRSV replication. These results indicate that the expression level of MARCO is closely related to the replication of PRRSV, and it is expected to become a new method for screening pigs resistant to blue ear disease, which has good clinical application value for the breeding of PRRSV resistant pigs.
  • the purpose of the present invention is to provide the application of MARCO in screening pigs resistant to blue ear disease.
  • Another object of the present invention is to provide a method for determining resistance or susceptibility of pigs to blue ear disease.
  • Yet another object of the present invention is to provide the application of MARCO in improving the resistance of pigs to blue ear virus, or the application of preparing medicines or preparations for improving the resistance of pigs to blue ear virus.
  • Another object of the present invention is to provide the application of MARCO in breeding pig breeds resistant to blue ear virus.
  • the research of the present invention finds that PRAMV infected PAMs cells in pigs inhibit MARCO expression at different time points and different infection complexes. That is, the expression of MARCO is closely related to PRRSV infection, and it is closely related to the susceptibility of pigs to blue ear disease. Moreover, interference of MARCO expression by siRNA will promote PRRSV replication, and MARCO overexpression inhibits PRRSV replication. That is, the higher the expression of MARCO in pigs, the more resistant they are to blue ear virus; otherwise, they are more susceptible.
  • the blue ear virus includes a classical strain and a highly pathogenic strain (HP-PRRSV).
  • MARCO expression activators such as overexpression
  • MARCO expression activator in the preparation of a medicament or a preparation for improving resistance to blue ear virus in pigs.
  • the present invention also provides a method for identifying the susceptibility of pigs to blue ear disease, specifically by detecting the MARCO expression level (expression amount) in pigs to identify whether pigs are resistant or susceptible to blue ear virus.
  • the identification criteria are: when MARCO expression is high, pigs are resistant to blue ear virus; otherwise, they are susceptible.
  • the level of MARCO expression is based on the general level of MARCO expression in pigs in the art. The high and low levels described here are relative. Based on whether the MARCO expression is high or low, it is determined whether the pig may be resistant to PRRSV and the degree of susceptibility to PRRSV.
  • the specific data criteria for identification are roughly: when the expression of MARCO is higher than 2.5 ⁇ 0.5 ⁇ 10 -2 (relative to the internal reference HPRT1), the pig is resistant to the virus; the expression is low Susceptible to 2.5 ⁇ 0.5 ⁇ 10 -2 (compared to internal reference HPRT1).
  • the method for detecting the expression level of MARCO in the present invention is not strictly limited, and may be an existing method for detecting the expression level of genes in the art.
  • the present invention provides a primer set for detecting the expression of porcine MARCO, including an upstream primer MARCO-F and a downstream primer MARCO-R, the sequences of which are shown in SEQ ID Nos. 1 to 2, respectively.
  • SEQ ID ID NO.1 5′-GCAGCGGGTAGACAACTTC-3 ′
  • the application of the primer set in judging the resistance or susceptibility of pigs to blue ear disease, or the application in screening pigs resistant to blue ear disease should also fall within the protection scope of the present invention.
  • the invention selects different breeds of pigs to study the application of MARCO in screening pigs resistant to blue ear disease.
  • the PRRSV challenge was selected from local breeds of Tongcheng and Meishan in China (not susceptible to blue ear disease) and foreign breeds of Changbai and Dabai pigs (susceptible to blue ear disease).
  • the virus titer TCID 50 and PRRSV N protein expression levels in lymph node tissues were used to determine the difference in PRRSV infectivity between Tongcheng pigs, Meishan pigs, and foreign breeds of Changbai and Dabai pigs. Based on the expression levels of MARCO, their PRRSV infectivity with 4 breeds of pigs was studied. relationship. The results show that the method for screening blue ear-resistant pigs using MARCO according to the present invention is reliable and accurate.
  • MARCO in pigs is closely related to PRRSV infection.
  • MARCO can be used as a new method to determine PRRSV resistance or susceptibility; when the expression of MARCO is abnormally high, pigs are resistant to PRRSV, and vice versa Susceptible.
  • the discovery of the present invention lays the foundation for screening of PRRSV-resistant pigs and PRRSV-resistant breeding. It has good clinical application value for the screening and breeding of PRRSV-resistant pigs, and is of great significance for the prevention and control of PRRSV. Conducive to the breeding of excellent strains of pigs. At the same time, the invention also provides a new application for MARCO.
  • Figure 1 shows the difference in the expression of MARCO in different tissues (lung, spleen, liver, kidney).
  • Figure 2 shows the expression levels of MARCO mRNA at different time points of PRRSV-infected PAMs cells.
  • Figure 3 shows the expression levels of MARCO protein at different time points of PRRSV-infected PAMs cells.
  • Figure 4 shows the expression levels of MARCO mRNA in different infection complexes of PRRSV-infected PAMs cells.
  • Figure 5 shows the MARCO mRNA expression level after MARCO interference by siRNA.
  • Figure 6 shows the mRNA expression level of PRRSV N protein after MARCO interference by siRNA.
  • Figure 7 shows PRRSV N protein expression levels after MARCO overexpression.
  • MYC is a MARCO label.
  • Figure 8 shows the mRNA expression level of PRRSV N protein after MARCO overexpression.
  • the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
  • the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
  • the recovered alveolar lavage fluid was gently blown back and forth with a pipette to separate the cell clumps.
  • the lavage fluid was centrifuged at 4 ° C and 1000 rpm for 10 minutes, and the supernatant was discarded.
  • the cells were resuspended in RPMI 1640 medium, centrifuged at 4 ° C, 1000 rpm for 5 min, and the supernatant was discarded.
  • Use RPMI-1640 nutrient solution containing 20% FBS to gently blow the resuspended cells.
  • add double antibodies divide the isolated cells evenly into the culture plate, and humidify the culture at 37 ° C and 5% CO 2 Incubator.
  • PAMs cells were cultured with RPMI 1640 medium containing 10% fetal bovine serum, PRRSV was inoculated with multiple infection multiple MOI, and cultured in RPMI 1640 medium with 2% fetal bovine serum for 24 hours at 37 ° C. Cells were collected. The genes were tested by qRT-PCR. The results are shown in Figure 4. After PRRSV infected PAMs cells, the expression of MARCO decreased significantly.
  • Reduced Serum Medium is diluted, mixed in a 1: 1 ratio and diluted at room temperature for 5 min, and then added to PAMs cells exchanged with fresh RPMI 1640 culture medium at 37 ° C, 5% CO 2 incubator for 6 h, discarding the supernatant, PBS Wash it once, continue to culture for 48 h with RPMI 1640 medium containing 10% fetal bovine serum, and collect cells to verify the MARCO interference effect by qRT-PCR experiments. After another 6-well plate was cultured for 48 hours, PRRSV was added to the culture medium and cultured for another 24 hours. Cells were collected and verified by qRT-PCR experiments for the effects of MARCO interference on PRRSV replication.
  • MARCO promotes PRRSV replication after siRNA interference.
  • MARCO overexpression inhibits PRRSV replication.
  • Tongcheng pig and Meishan pig are not susceptible to blue ear virus, that is, they are resistant; and Changbai pig and big white pig are more susceptible to blue ear virus.
  • Tongcheng pigs, Meishan pigs, long white pigs and big white pigs were selected. Each 3 pigs were divided into two groups. One group was subjected to PRRSV challenge, and the other group was not subjected to any treatment (control). Blood was collected on the 1st day before the poisoning and on the 7, 14, 21, and 28th days after the challenge. The blood was collected from the control group at the same time, and the lungs and lymph node tissues were collected on the 28th day.
  • RNA Take a part of the serum for TCID 50 measurement, extract RNA from the remaining serum and lung and lymph node tissues, invert, perform qRT-PCR detection of MARCO and PRRSV N proteins, and make pathological sections of lung tissue.
  • PRRSV N protein expression levels and lung tissue pathological sections, Tongcheng pigs, Meishan pigs and foreign breeds of Changbai pigs and Dabai pigs were determined for PRRSV infectivity, and their relationship with 4 breeds of pigs was measured according to MARCO expression levels. .
  • the specific data standard for identifying pigs' susceptibility to blue ear virus is roughly: when the expression of MARCO is higher than 2.5 ⁇ 0.5 ⁇ 10 -2 (compared with the internal reference HPRT1), pigs are resistant to the virus sexuality; susceptibility when the expression level is less than 2.5 ⁇ 0.5 ⁇ 10 -2 (relative to the internal reference HPRT1).

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Abstract

本发明公开了MARCO在筛选抗蓝耳病猪中的应用。本发明研究显示，猪体内MARCO表达量与PRRSV感染息息相关，PRRSV感染PAMs后抑制MARCO表达，MARCO干扰促进PRRSV复制，MARCO过表达抑制病毒复制；当体内MARCO表达量异常较高时，猪对PRRSV具有抗性，反之易感。因此，MARCO可以作为一种新型的判定PRRSV易感性或抗性的标准，为鉴别猪对PRRSV易感性以及PRRSV抗性猪的筛选和培育奠定了坚实的基础，对PRRSV抗性猪的筛选和培育具有很好的临床应用价值，对猪蓝耳病的防控具有重要的意义，也有利于培育优良品系猪。

Description

MARCO在筛选抗蓝耳病猪中的应用 技术领域
本发明属于生物医药技术领域。更具体地，涉及MARCO在筛选抗蓝耳病猪中的应用。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome，PRRS)，俗称蓝耳病，是由猪繁殖与呼吸综合征病毒(Porcine reproductive andrespiratory syndrome virus，PRRSV)引起。早在1992年，世界动物卫生组织(Office International Des Epizooties，OIE)就将该病列为B类传染病。目前，PRRS是养猪业中最重要的传染病之一，几乎分布在所有的养猪国家，在全球对养猪业造成巨大的经济损失。2006年，我国大部分省份及越南爆发了猪高热病(Porcine high fever syndrome，PHFS)，给养猪业造成了巨大的经济损失，此病是由变异的PRRSV引起，命名为高致病型猪繁殖与呼吸综合征病毒(Highly pathogenic porcine reproductive and respiratory syndrome virus，HP-PRRSV)。HP-PRRSV特征为高热、高致病性及高死亡率。目前，农业部已将该病列入重大动物疫病强制免疫计划。
PRRSV的防控是目前我国乃至世界的难题。PRRSV难于防控主要表现在以下几方面：(1)嗜巨噬细胞性和免疫抑制性疾病，PRRSV主要感染猪的肺泡巨噬细胞(Porcine alveolar macrophages，PAMs)，PAMs是免疫细胞，破坏PAMs，从而破坏机体免疫系统，从而引起免疫抑制；(2)抗原变异性，目前PRRSV变异较快，弱毒疫苗的使用是促使病毒变异的一个原因，最近有文献报道，美国出现了蓝耳新毒株NADC30，而我国也分离到类似美国的新毒株，命名为NADC30-like，而另有文献报道从猪场中分离到与疫苗毒基因组高度同源的PRRSV致病毒株，且毒力增强，分析有可能是疫苗毒反强或重组并散毒；(3)疫苗没有交叉保护力，目前市场上PRRSV疫苗几乎无交叉保护力，不同毒株间不交叉保护力；(4)抗体依赖性增强，PRRSV的感染会刺激机体产生抗体，但高效价的抗体不但不能中和病毒，反而对病毒的增殖有促进作用；(5)病毒持续性感染，PRRSV感染后，能够长时间在猪体内检测到病毒血症，PRRSV在体内 持续时间长达5个月；(6)混合感染，目前临床上多见蓝耳与其他疾病混合感染，特别是圆环病毒、副猪嗜血杆菌、猪肺疫等与PRRSV的混合感染，使PRRSV的防控难上加难。
另外，由于遗传背景与生物抗病性有着紧密的联系，因此，基于基因手段筛选蓝耳病抗性猪的工作，对于该疾病的防控具有不可比拟的优势，重点在于找出抗性或易感相关的基因，不仅可以研究蓝耳病的感染机制，还为蓝耳病的治疗和预防提供新办法。
发明内容
本发明要解决的技术问题是克服现有猪蓝耳病防治技术的缺陷和不足，提供一种与猪蓝耳病抗性相关基因，本发明研究表明，PRRSV感染PAMs细胞后抑制MARCO表达，MARCO过表达抑制PRRSV复制，MARCO干扰促进PRRSV复制。表明MARCO的表达量与PRRSV的复制息息相关，有望成为一种新型的筛选抗蓝耳病猪的方法，对PRRSV抗性猪的培育具有很好的临床应用价值。
本发明的目的是提供MARCO在筛选抗蓝耳病猪中的应用。
本发明的另一目的是提供一种判定猪对蓝耳病抗性或易感性的方法。
本发明的再一目的是提供MARCO在提高猪对蓝耳病毒的抗性方面的应用，或在制备提高猪对蓝耳病毒抗性的药物或制剂方面的应用。
本发明的再一目的是提供MARCO在培育对蓝耳病毒具有抗性的猪品种方面的应用。
本发明上述目的通过以下技术方案实现：
本发明研究发现，猪体内PRRSV感染PAMs细胞不同时间点、不同感染复数均抑制MARCO表达。即MARCO表达量与PRRSV感染息息相关，与猪对蓝耳病易感性有密切的关系，且经siRNA干扰MARCO的表达后会促进PRRSV复制，MARCO过表达后抑制PRRSV复制。即猪体内MARCO表达量较高，则其对蓝耳病毒具有抗性；反之易感性更高。
因此，以下应用均应在本发明的保护范围之内：
MARCO在筛选抗蓝耳病猪中的应用。
所述猪MARCO的Gene ID：100516298。
所述蓝耳病毒包括经典毒株和高致病性毒株(HP-PRRSV)。
MARCO在提高猪对蓝耳病毒的抗性方面的应用。
MARCO在制备提高猪对蓝耳病毒抗性的药物或制剂方面的应用。
MARCO在筛选和/或培育对蓝耳病毒具有抗性的猪品种方面的应用。
MARCO表达激活剂(如过表达)在提高猪对蓝耳病毒抗性方面的应用。
MARCO表达激活剂在制备提高猪对蓝耳病毒抗性的药物或制剂方面的应用。
另外，本发明还提供一种鉴别猪对蓝耳病易感性的方法，具体是通过检测猪体内MARCO表达水平(表达量)来鉴别猪对蓝耳病毒是抗性或易感性。
鉴别的标准是：MARCO表达量高时，猪对蓝耳病毒具有抗性；反之具有易感性。
具体操作时，MARCO表达量的高低以本领域技术中猪体内MARCO表达量的普遍水平为依据。这里所述的高低是相对的，根据MARCO表达量较高或较低异常，从而判定猪是否可能对PRRSV具有抗性，对PRRSV的易感性程度。
更具体地，基于本发明实验的结果，鉴别的具体数据标准大致为：当MARCO表达量高于2.5±0.5×10 -2(相对于内参HPRT1)时，猪对病毒有抗性；表达量低于2.5±0.5×10 -2(相对于内参HPRT1)时，易感。
本发明对于MARCO表达量的检测方法不作严格限制，可以是本领域内现有的基因表达量检测方法。
作为一种可优选的实施方案，本发明提供了一种检测猪MARCO表达量的引物组，包括上游引物MARCO-F和下游引物MARCO-R，序列分别如SEQ ID NO.1～2所示。
SEQ ID NO.1所示序列：5′-GCAGCGGGTAGACAACTTC-3′，
SEQ ID NO.2所示序列：5′-TGTTGCTCCATCTTGTCCCG-3′。
所述引物组在判定猪对蓝耳病抗性或易感性方面的应用，或在筛选抗蓝耳病猪中的应用，也都应在本发明的保护范围之内。
本发明选用了不同品种猪研究MARCO在筛选抗蓝耳病猪中的应用。分别选取我国地方品种通城猪、梅山猪(对蓝耳病不易感)及国外品种长白猪、大白猪(对蓝耳病易感)为例，进行PRRSV攻毒，解剖后根据血清和肺、淋巴结组织中病毒滴度TCID 50及PRRSV N蛋白表达水平确定通城猪、梅山猪及国外品种长白猪、大白猪对PRRSV感染力差别，根据MARCO表达水平研究其与4种品种猪对PRRSV感染力关系。结果显示，本发明所述利用MARCO筛选抗蓝耳病 猪的方法是可靠的、准确的。
本发明具有以下有益效果：
本发明首次研究发现了猪体内MARCO表达量与PRRSV感染息息相关，MARCO可作为一种新型的判定PRRSV抗性或易感性的方法；当MARCO表达量异常较高时，猪对PRRSV具有抗性，反之易感。
本发明的发现为筛选抗蓝耳病猪以及PRRSV抗病育种奠定基础，对PRRSV抗性猪的筛选和培育具有很好的临床应用价值，对猪蓝耳病的防控具有重要的意义，也有利于优良品系猪的培育。同时，本发明也为MARCO提供了一种新的应用。
附图说明
图1为MARCO在不同组织(肺、脾脏、肝脏、肾脏)中表达量差异。
图2为PRRSV感染PAMs细胞不同时间点MARCO mRNA表达水平。
图3为PRRSV感染PAMs细胞不同时间点MARCO蛋白表达水平。
图4为PRRSV感染PAMs细胞不同感染复数MARCO mRNA表达水平。
图5为MARCO经siRNA干扰后，MARCO mRNA表达水平。
图6为MARCO经siRNA干扰后，PRRSV N蛋白mRNA表达水平。
图7为MARCO过表达后，PRRSV N蛋白表达水平。MYC为MARCO标签。
图8为MARCO过表达后，PRRSV N蛋白mRNA表达水平。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明以下实施例的统计学分析：所有试验至少3次独立重复，结果采用平均值和标准误表示，使用单因素方差分析和T检测分析。所有统计分析均采用以P<0.05作为具有显著统计学差异的检验标准，分析软件为SPSS 16.0和GraphPad Prism 5。
实施例1 PAMs细胞分离和培养
1、经检测无PRRSV、猪圆环病毒2型(PCV-2)、猪瘟病毒(CSFV)等特 定病原感染的SPF级6-8周龄的断奶仔猪绑定后，前腔静脉放血致死，结扎气管，剖开胸部，连同心脏取出完整的肺脏，用无菌PBS液充分漂洗肺表面，清除血块和污物，清除完毕后摘除心脏。灌入PBS缓冲液，轻轻拍打捏揉肺表面5-10min，回收支气管肺泡灌洗液。重复步骤2至3次，直至共回收约1000mL灌洗液。将回收得到的肺泡灌洗液用移液器轻轻来回吹打，使细胞团块分开，4℃，1000rpm离心灌洗液10min，弃去上清。RPMI 1640培养基重悬细胞，4℃，1000rpm离心5min，弃去上清。用含20％的FBS的RPMI-1640营养液轻轻吹打重悬细胞，为防止操作污染，加入双抗，将分离得到的细胞均匀分到培养板中，于37℃、5％CO 2加湿培养箱中培养。
2、观察PAMs细胞形态、活性，液氮保存备用。
实施例2 MARCO基因体外调节PRRSV复制的研究
1、用含10％胎牛血清的RPMI 1640培养液培养PAMs细胞，以MOI＝0.1接毒PRRSV，在2％胎牛血清的RPMI 1640培养液中37℃继续培养，培养不同时间点收集细胞，分别对MARCO基因做qRT-PCR和Western-blot检测。结果如图2、图3所示，PRRSV感染PAMs细胞后，MARCO表达量显著降低。
2、用含10％胎牛血清的RPMI 1640培养液培养PAMs细胞，以不同感染复数MOI接毒PRRSV，在2％胎牛血清的RPMI 1640培养液中37℃继续培养24h，收集细胞，对MARCO基因做qRT-PCR检测。结果如图4所示，PRRSV感染PAMs细胞后，MARCO表达量显著降低。
实施例3 MARCO干扰
1、将设计好的MARCO siRNA及1对Negative Control(对照)送Thermo Fisher Scientific公司合成，使用Lipofectamine TMRNAiMAX转染试剂(购自Thermo Fisher Scientific公司)。在2个6孔板中用含10％胎牛血清的RPMI 1640培养液培养PAMs细胞至60-70％密度，分别将siRNA(3对siRNA和1个对照)及Lipofectamine TMRNAiMAX用
Figure PCTCN2018099287-appb-000001
Reduced Serum Medium稀释，稀释后按1:1比例混合并室温培养5min，然后加入到换有新鲜RPMI 1640培养液的PAMs细胞上37℃、5％CO 2培养箱中培养6h，弃上清，PBS洗1遍，用含10％胎牛血清的RPMI 1640培养液继续培养48h，收集细胞通过qRT-PCR实验验证MARCO干扰效果。另外一个6孔板培养48h后，在培养基中加入PRRSV，再培养24h，收集细胞通过qRT-PCR实验验证MARCO干扰后对PRRSV复制影 响。
2、结果如图5所示，MARCO经siRNA干扰后，相对于对照组，siRNA显著地抑制了MARCO mRNA表达。
MARCO干扰后，PRRSV接毒，结果如图6所示，相对于对照组，siRNA处理后，PRRSV N蛋白mRNA水平显著升高。
综上所述，MARCO经siRNA干扰后促进PRRSV复制。
实施例4 MARCO过表达
1、构建pcDNA3.1-MARCO过表达载体，在2个6孔板中用含10％胎牛血清的RPMI 1640培养液培养PAMs细胞至60-70％密度，使用Lipofectamine TM2000(购自Thermo Fisher Scientific公司)分别将pcDNA3.1-MARCO和pcDNA3.1空载体转入Marc-145细胞，在37℃、5％CO 2培养箱中培养6h，弃上清，PBS洗1遍，用含10％胎牛血清的RPMI 1640培养液继续培养48h，收集细胞检测过表达效果。另外一个6孔板培养48h后，在培养基中加入PRRSV，再培养24h，收集细胞检测MARCO过表达后对PRRSV复制影响。
2、结果如图7所示，MARCO过表达后，MYC有表达，表明MARCO过表达成功；同时，显著地抑制了PRRSV N蛋白表达。
MARCO过表达后，PRRSV接毒，结果如图8所示，相对于对照组，PRRSV N蛋白mRNA水平显著降低。
综上所述，MARCO过表达后抑制PRRSV复制。
实施例5 MARCO在筛选抗蓝耳病猪中的应用
1、选取我国地方品种通城猪、梅山猪及国外品种长白猪、大白猪作为实验对象，验证本发明利用MARCO筛选抗蓝耳病猪的方法。
其中，已知通城猪和梅山猪对蓝耳病毒不易感，即具有抗性；而长白猪和大白猪对蓝耳病毒更易感。
2、实验方法
选择通城猪、梅山猪、长白猪、大白猪各6头，每3头1组，即分成2组，其中1组进行PRRSV攻毒，另外1组不做任何处理(对照)，分别在攻毒前1d和攻毒后第7、14、21、28d采血，对照组同步采血，第28d处死，采集肺、淋巴结组织。
取一部分血清进行TCID 50测定，剩余血清及肺、淋巴结组织提取RNA、反 转，对MARCO及PRRSV N蛋白进行qRT-PCR检测，并对肺组织做病理切片。
根据TCID 50、PRRSV N蛋白表达水平及肺组织病理切片确定通城猪、梅山猪及国外品种长白猪、大白猪对PRRSV感染力，根据MARCO表达水平研究其与4种品种猪对PRRSV感染力关系。
3、根据结果可判断，通城猪和梅山猪对蓝耳病毒不易感，而长白猪和大白猪对蓝耳病毒更易感。
同时，MARCO表达水平检测结果显示，通城猪和梅山猪体内MARCO表达水平显著高于长白猪和大白猪。
另外，基于大量实验结果显示，鉴别猪对蓝耳病毒感病性的具体数据标准大致为：当MARCO表达量高于2.5±0.5×10 -2(相对于内参HPRT1)时，猪对病毒有抗性；表达量低于2.5±0.5×10 -2(相对于内参HPRT1)时，易感。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. MARCO在筛选抗蓝耳病猪方面的应用。
  2. 一种鉴别猪对蓝耳病易感性的方法，其特征在于，是通过检测猪体内MARCO表达水平来鉴别猪对蓝耳病毒是抗性或易感性。
  3. 根据权利要求2所述的方法，其特征在于，鉴别的标准是：MARCO表达量高时，猪对蓝耳病毒具有抗性；反之具有易感性。
  4. 一种检测猪MARCO表达量的引物组，其特征在于，所述引物组包括上游引物MARCO-F和下游引物MARCO-R，序列分别如SEQ ID NO.1～2所示。
  5. 权利要求4所述引物组在判定猪对蓝耳病抗性或易感性方面的应用。
  6. 权利要求4所述引物组在筛选抗蓝耳病猪中的应用。
  7. MARCO在提高猪对蓝耳病毒的抗性方面的应用。
  8. MARCO在制备提高猪对蓝耳病毒抗性的药物或制剂方面的应用。
  9. MARCO在培育对蓝耳病毒具有抗性的猪品种方面的应用。
  10. MARCO表达激活剂在提高猪对蓝耳病毒抗性方面的应用，或在制备提高猪对蓝耳病毒抗性的药物或制剂方面的应用。
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