CN111202839A - 一种组蛋白h4在抑制禽流感病毒中的应用 - Google Patents
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Abstract
本公开提供了一种组蛋白H4在抑制禽流感病毒中的应用，所述禽流感病毒为H9N2亚型禽流感病毒。所述H9N2亚型禽流感病毒经所述组蛋白H4在体外作用后接种SPF鸡胚，鸡胚死亡数量明显下降，胚液HA效价也明显降低，即组蛋白H4能够有效的抑制H9N2亚型禽流感病毒。
Description
技术领域
本公开属于动物免疫技术领域和生物医药技术领域，具体涉及一种组蛋白H4在抑制禽流感病毒中的应用。
背景技术
H9N2型禽流感其病毒(AIV)属于正粘病毒科、正粘病毒属；病毒粒子多呈球形，为80～120纳米的直径，表面有长10～12纳米的密集钉状物或纤突覆盖，包括血凝素(HA)和神经氨酸酶(NA)两种不同形状的表面钉状物，病毒囊膜内有螺旋形核衣壳。H9N2型禽流感给养鸡业带来巨大损失，该病不但造成鸡呼吸道病，还侵害鸡的生殖系统，造成肉鸡生长缓慢，蛋鸡产蛋下降，于此同时还易与其他病原微生物混合感染，可以造成严重的死亡。
组蛋白(histone)是真核生物体细胞染色质与原核细胞中的碱性蛋白质，和DNA共同组成核小体结构，含有102至135个氨基酸，由三个二环连接的a-螺旋结构折叠而成。它们是染色质的主要蛋白质组分，作为DNA缠绕的线轴，并在基因调控中发挥作用。组蛋白通常含有H1、H2A、H2B、H3和H4等5种成分，其中组蛋白H3和H4结合形成H3-H4二聚体，两个H3-H4二聚体结合形成四聚体，该四聚体进一步与两个H2a-H2b二聚体组合形成致密组蛋白八聚体核心，H4有一个球状结构和一条长尾N末，组蛋白是高度转录后修饰的，共价结合修饰包括N末端的乙酰化和甲基化，这些修饰可能会改变其亲代组蛋白八聚体相关的基因的表达。组蛋白H4是染色质结构和功能的重要蛋白，相关研究认为其序列变异和共价修饰在基因调控中起着重要作用。目前已发现组蛋白H2A、H2B、H3和H4具有治疗利什曼病的作用，但是目前还未出现关于组蛋白在治疗或抑制禽流感病毒上的研究报道。
发明内容
本公开的目的是提供一种组蛋白H4在抑制禽流感病毒中的应用，以达到抑制禽流感，减少养殖业经济损失的目的。
为实现上述目的，技术方案如下：
一种组蛋白H4在抑制禽流感病毒中的应用，所述禽流感病毒为H9N2。
所述H4组蛋白的核苷酸序列如SEQ ID NO.1所示。
所述H4组蛋白的氨基酸序列如SEQ ID NO.2所示。
本公开的有益效果是：提供了一种组蛋白H4在抑制禽流感病毒中的应用，所述禽流感病毒为H9N2亚型禽流感病毒，所述H9N2亚型禽流感病毒经所述组蛋白H4在体外作用后接种SPF鸡胚，鸡胚死亡数量明显下降，胚液HA效价也明显降低，即组蛋白H4能够有效的抑制H9N2亚型禽流感病毒。
具体实施方式
以下各步骤仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各步骤对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各步骤所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各步骤技术方案的范围。
实施例1
一种组蛋白H4在抑制禽流感病毒中的应用
1.材料
组蛋白H4储存液：根据组蛋白H4的核苷酸序列如SEQ ID NO.1所示，经生物公司合成，得到组蛋白H4，其中组蛋白H4的氨基酸序列如SEQ ID NO.2所示。
组蛋白H4工作液：每管组蛋白H4储存液(11μL)中加入348μL生理盐水。
禽流感H9N2亚型病毒株(AIV)；9日龄SPF鸡胚。
2.实验方法
实验组：将纯化的禽流感H9N2亚型病毒液1.5μL、生理盐水934.5μL和组蛋白H4工作液264μL进行混合，将禽流感H9N2亚型病毒稀释800倍；然后将混合液置于37℃培养箱中进行孵育，其中孵育的时间为2h。
阳性对照组：将纯化的禽流感H9N2亚型病毒液15μL与11985μL的生理盐水进行混合，然后将混合液置于37℃培养箱中进行孵育，其中孵育的时间为2h。
将上述孵育后的实验组的混合液接种至9日龄SPF鸡胚尿囊腔中，共15组重复，其中每组接种的混合液为0.2mL；然后设置阳性对照组：将上述阳性对照组中的混合液接种至9日龄SPF鸡胚尿囊腔中，共15组重复，接种完毕后，将所有鸡胚置于37℃孵化箱中继续培养，每天观察2次接种鸡胚的死亡情况，并及时收集死亡接种鸡胚的尿囊液，观察鸡胚胚体病变情况，在接种5天后，将存活的接种鸡胚置于4℃冰箱中进行冷冻处死，然后收集鸡胚尿囊液。
因为禽流感H9N2亚型病毒(AIV)具有凝集鸡红细胞的特性，因此可通过对禽流感H9N2亚型病毒(AIV)中HA的血凝活，来判断感染情况，其中HA效价≥4为感染。
其中HA血凝实验具体操作步骤为：
(1)首先在96孔微量反应板上各孔加入50μL的生理盐水；
(2)于左侧第1孔加50μL鸡胚尿囊液，混合均匀后，吸50μL至第2孔，依次倍比稀释至第11孔，吸弃50μL；
(3)依次向各孔加入0.5％鸡红细胞悬液50μL，在振荡器上振荡，37℃静置30min后观察结果。
3.结果分析
观察并记录接种后鸡胚的死亡情况和并检测血凝活性，结果如表1所示，从表1可以看出，组蛋白中的H4在体外作用的H9N2亚型禽流感病毒接种SPF鸡胚，鸡胚死亡数量明显下降，胚液HA效价也明显降低，即组蛋白H4能够有效的抑制H9N2禽流感病毒。
表1组蛋白H4与流感病毒H9N2反应后鸡胚死亡情况表
SEQUENCE LISTING
<110> 佛山科学技术学院
<120> 一种组蛋白H4在抑制禽流感病毒中的应用
<130> 2020.02.19
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 715
<212> DNA
<213> 人工合成
<400> 1
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aaaggaggaa aagggttagg caaaggagga gcaaagagac acagaaaggt tctaagagac 180
aacattcaag gaatcacaaa gccagcgatt cgtcgtcttg ctcgtagagg aggtgtgaag 240
agaatcagtg gattgatcta tgaagaaacg agaggtgtgt tgaagatttt tctggagaat 300
gtgattagag atgctgttac ttacactgag catgcgagga ggaagacggt gactgctatg 360
gatgttgttt atgccttgaa gagacaagga agaactctat atggatttgg tggttgatca 420
atttgagatc tgggttttct ggtgaatgat gatgatttaa gtcttgcgat caagaaattc 480
cagaaattgg gttgaatttt agggtttcgt tttgtgttgt aattagggca gcattgtaat 540
ggattaatga taagtaccat ttgctctaat tactctttaa tctctgaaat tcatggtaaa 600
ggattatcaa tcgaaaacta atcaaaggaa ttgattgaac tatgtttttg aagattgaaa 660
aacaaataag gactaaatgt gagcaattta aagtttagag gtataagaat cgaat 715
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Met Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala
1 5 10 15
Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys
20 25 30
Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser
35 40 45
Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu
50 55 60
Asn Val Ile Arg Asp Ala Val Thr Tyr Thr Glu His Ala Lys Arg Lys
65 70 75 80
Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln Gly Arg
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Thr Leu Tyr Gly Phe Gly Gly
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Claims (3)
1.一种组蛋白H4在抑制禽流感病毒中的应用，其特征在于，所述禽流感病毒为H9N2亚型禽流感病毒。
2.根据权利要求书1中所述组蛋白在抑制禽流感病毒中的应用，其特征在于，所述H4组蛋白的核苷酸序列如SEQ ID NO.1所示。
3.根据权利要求书1中所述组蛋白在抑制禽流感病毒中的应用，其特征在于，所述H4组蛋白的氨基酸序列如SEQ ID NO.2所示。
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