CN116396400A - 一种禽白血病病毒多亚型抗原表位融合蛋白及其应用 - Google Patents
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Abstract
本发明提供一种禽白血病病毒多亚型抗原表位融合蛋白、多亚型识别抗体、检测试剂盒及其应用。本发明所述禽白血病病毒多亚型抗原表位融合蛋白其氨基酸序列如SEQ ID NO:1所示。实验表明,该禽白血病病毒多亚型抗原表位融合蛋白对禽白血病病毒多亚型血清有反应同时对其他常见鸡感染病毒或细菌血清无反应。本发明所述禽白血病病毒多亚型抗原表位融合蛋白免疫动物可制备多亚型识别抗体。本发明所述多亚型识别抗体可以与多亚群的禽白血病特异性抗原表位特异性识别,开发检测试剂盒,用于鸡种群禽白血病病毒的快速筛选和净化,具有良好的应用前景。
Description
技术领域
本发明属于生物医药制剂领域,具体是一种禽白血病病毒抗原表位融合蛋白及其应用。
背景技术
禽白血病是由反转录病毒科α反转录病毒属成员禽白血病病毒(Avian leucosisviruse,ALV)引起的禽类多种肿瘤性疾病的统称。根据禽白血病病毒的囊膜糖蛋白特异性及宿主范围和病毒的交叉可以分为11个亚群(A-K),其中A-J和K亚群主要对鸡致病。其中A、B和J亚群为发生于田间鸡的外源性病毒。J亚型ALV可水平传播和垂直传播,垂直传递可导致子代免疫耐受,抗体检测表现为阴性。同时发病鸡出现急性肿瘤引起死亡,一旦发现患病需立即处理。AB亚型ALV主要通过垂直传播影响鸡雏质量,病鸡体重增长缓慢,产蛋量减少。K亚型病毒在不同鸡群中被发现,表明K亚群病毒在鸡群中的感染呈增长的趋势,可能成为继J亚群禽白血病病毒之后成为我国养鸡业新的重大潜在威胁。
本病即可水平传播也可垂直传播,先天感染的免疫耐受鸡是最重要的传染源,在配种环节的交叉污染是病原在鸡群间水平传播的一大途径。该病没有可用的疫苗,鸡群净化是目前防止感染的唯一途径。虽然现有技术中针对ALV的某一个亚群的检测相对成熟,实现了对不同亚群的区分,但是这种区分在种群的净化过程中并没有太大意义。虽然检测结果显示不含有其中的某一亚群,但并不能排除其他亚群的感染。因此迫切的需要一种检测手段对ALV进行检测,这个过程中并不需要区分到底是哪个亚型,而是通过结果可以快速准确的一票否决,实现对鸡种群的快速筛选和净化。
发明内容
本发明目的在于提供一种包含禽白血病病毒多亚型抗原表位融合蛋白,所述融合蛋白中含有A、B、J、K亚群的特异性识别抗原表位,可以同时实现对常见ALV的快速筛选。
本申请所述的融合蛋白的氨基酸序列为RKLLVSCLKLGGGSSPRSDWSIDTLSRRYCGNEFTSARGGSSDILKSRTILANSGICFQDCLSRTGGSQSREINETEPFS FMNLVLCVTGEV(SEQ ID NO.1)。
进一步的,本发明提供编码上述融合蛋白的DNA分子。由于密码子的简并性,可以存在很多种能够编码本发明所述的特异性抗原表位的核苷酸序列。
本发明还提供了一种重组表达载体,含有本发明所述的DNA分子。
另一方面,本发明还提供了一种工程菌,其含有本发明所述的重组表达载体。
在本发明中,“工程菌”通过将重组表达载体热激转化进入宿主细胞所获得。本发明中所表达的“宿主细胞”包括原核或真核细胞。
所述宿主细胞选自大肠杆菌、酵母、昆虫或哺乳动物细胞。
本发明还要求保护禽白血病病毒的多亚型识别抗体,其是由所述融合蛋白免疫动物制备而成的单克隆抗体或多克隆抗体。
本发明所述多亚型识别抗体可以与A、B、J、K亚群禽白血病发生特异性识别,与其他的常见鸡群病毒或者细菌并没有交叉反应。
进一步的,本发明提供一种检测试剂盒,用于鸡群的快速净化。
进一步的,所述检测试剂盒含有:
(1)本发明所述的禽白血病病毒多亚型抗原表位融合蛋白;
和/或
(2)本发明所述的多亚型识别抗体。
优选的,所述的多亚型识别抗体可以为单克隆抗体,也可以为多克隆抗体。
优选的,本发明所述的试剂盒中还包括酶标板。
本领域技术人员可以理解,本发明提供的试剂盒可将禽白血病病毒多亚型抗原表位融合蛋白固定于酶标板用于禽白血病病毒抗体的检测,也可将抗体包被于酶标板,用于检测禽白血病病毒。
进一步的,作为优选,本发明所述试剂盒还包括包被液、封闭液、二抗、显色液、终止液。
进一步的,本发明提供一种禽白血病病毒多亚型抗原表位融合蛋白、多亚型识别抗体、检测试剂盒的应用,所述应用为鸡种群禽白血病病毒的快速筛选和净化。
优选的,所述的鸡种群禽白血病病毒的快速筛选和净化是非诊断目的以及非治疗目的的。
有益效果
本发明提供了一种禽白血病病毒多亚型抗原表位融合蛋白、多亚型识别抗体、检测试剂盒及其应用。本发明所述禽白血病病毒多亚型抗原表位融合蛋白其氨基酸序列如SEQ ID NO:1所示。实验表明,该禽白血病病毒多亚型抗原表位融合蛋白对禽白血病病毒多亚型血清有反应同时对其他常见鸡感染病毒或细菌血清无反应。本发明所述禽白血病病毒多亚型抗原表位融合蛋白免疫动物可制备多亚型识别抗体。本发明所述多亚型识别抗体可以与多亚群的禽白血病特异性抗原表位特异性识别,开发检测试剂盒,用于鸡种群禽白血病病毒的快速筛选和净化,具有良好的应用前景。
附图说明
图1为本发明禽白血病病毒多亚型抗原表位融合蛋白的高级结构预测。
图2为检测抗原表位的特异性和敏感度检测结果
具体实施方式
除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合具体实施例对本发明作进一步的详细说明。
实施例1抗原表位融合多肽的筛选
首先针对近年来我国较为流行的ALV-A,ALV-B,ALV-J,ALV-K病毒的gp85蛋白、p27蛋白的氨基酸序列用ClustalX2结合MEGA5.1软件进行初步分析,筛选获得了12个多肽候选序列,并利用生物分析手段筛选抗原片段候选区域,并根据生物信息学分析对其中的部分氨基酸残基进行替换或者优化,并利用柔性linker或者直接连接的方式对高级空间结构进行微调(参见图1),最终确定其抗原表位融合多肽的氨基酸序列为RKLLVSCLKLGGGSSPRSDWSIDTLSRRYCGNEFTSARGGSSDILKSRTILANSGICFQDCLSRTGGSQSREINETEPFS FMNLVLCVTGEV(SEQID NO.1)。通过多肽固相合成仪合成,并进行脱盐初步纯化(华大基因)。
利用overlap PCR方法构建抗原表位融合多肽表达盒,利用双酶切将其连接到克隆至PET-28a(购自Invitrogen公司,货号A11499),挑取单克隆鉴定插入方向,插入方向正确的质粒送Invitrogen公司测序,测序正确的质粒命名为PET-28a-ALV。将PET-28a-ALV重组阳性质粒进行表达,经SDS-PAGE验证后经镍柱层析法纯化获得可溶性融合蛋白。
实施例2、检测抗原表位的特异性和敏感度检测
用ALV的A、B、J、K亚群、新城疫病病毒(NDV)、禽流感病毒(AIV)H5亚型、鸡传染性法氏囊病病毒(IBDV)、鸡网状内皮组织增生症病毒(REV)、鸡柔嫩艾美耳球虫、霍乱弧菌(Vibrio cholerae)的阳性血清对筛选出的融合蛋白进行鉴定,用间接ElISA法检测融合蛋白效果。具体检测方法为:取合成的融合蛋白用DMSO重悬至20mg/ml,分别用pH9.6碳酸盐缓冲液(包被液)稀释至20μg/ml包被CORNING酶标板,4℃过夜,未包被组作为对照用于阴阳性判定标准;加2%脱脂乳37℃封闭2h;每孔加300μlPBST清洗,每次倒出液体时甩干酶标板中残留水分并在干净的吸水纸上控干,清洗5遍;加上述各种病毒/细菌的阳性血清或阴性对照100μl/孔,血清稀释倍数为50倍、250倍、1000倍,37℃孵育1小时,清洗5遍;加1:5000倍稀释的辣根过氧化物酶标记的羊抗鸡IgY(IgG)酶标二抗100μl/孔,37℃孵育1小时,清洗5遍;加TMB显色剂100μl/孔,静止10-15分钟,加终止液50μl后酶标仪检测D450值。结果的判定由检测OD450>标准阴性样品的均值+2×标准阴性样品的标准差进行阳性判定,在统计学上该判定结果可以达到95%的置信区间。
结果显示本发明筛选获得的禽白血病病毒多亚型抗原表位融合蛋白对ALV的A、B、J、K亚群都有明显的阳性反应,而对新城疫病病毒(NDV)、禽流感病毒(AIV)H5亚型、鸡传染性法氏囊病病毒(IBDV)、鸡网状内皮组织增生症病毒(REV)、鸡柔嫩艾美耳球虫、霍乱弧菌(Vibrio cholerae)的阳性血清基本无反应,结果证实本发明筛选获得的禽白血病病毒多亚型抗原表位融合蛋白具有较高的特异性和敏感性。(图2)
实施例3禽白血病病毒多亚型抗原表位融合蛋白特异性抗体的制备
选用体重约2kg健康雌性大耳白家兔(购自中国人民解放军军事医学科学院动物中心,实验前测定家兔的自然抗体,有自然抗体者淘汰),首次用福氏完全佐剂与实施例1获得的禽白血病病毒多亚型抗原表位融合蛋白混合乳化后皮下多点注射,然后分别于初次免疫后第20日、第30日、第40日追加免疫水剂1针,追加剂量和途径与初次免疫相同,末次免疫后10天试血,用玻片凝集试验检测血清效价,结果效价大于1∶640,颈动脉采血,离心收集抗血清。采用常规的饱和硫酸铵盐析法(33%饱和硫酸铵盐析3次,透析脱盐后收集抗体)对抗血清中的禽白血病病毒多亚型抗原表位融合蛋白特异性抗体进行纯化,得到禽白血病病毒多亚型抗原表位融合蛋白特异性抗体。采用紫外分光光度仪测定抗体浓度,方法:将纯化抗体适当稀释后,分别在280nm及260nm波长下测定A值,代入公式:蛋白含量(mg/mL)=(1.45×A280nm-0.74×A260nm)×稀释倍数,计算得到蛋白的浓度。结果按照上述方法纯化所得的抗体蛋白浓度约为10mg/mL。
实施例4抗体特异性的检测
利用间接ELISA法,包被原分别为ALV的A、B、J、K亚群的gp85蛋白,新城疫病病毒(NDV)的HN抗原、禽流感病毒(AIV)H5亚型非结构蛋白NS1、鸡传染性法氏囊病病毒(IBDV)的VP2抗原、鸡网状内皮组织增生症病毒(REV)囊膜蛋白、鸡柔嫩艾美耳球虫破碎液、霍乱弧菌(Vibrio cholerae)培养液,100ng/孔,对该多亚群抗体进行检测(表1),结果显示上述抗体对ALV的A、B、J、K亚群反应都为阳性,但对其他病毒、细菌或寄生虫的检测结果均为阴性,确定该抗体为禽白血病病毒多亚型特异性抗体。
表1抗体型别特异性的检测结果
检测对象 | 结果 | 检测对象 | 结果 | 检测对象 | 结果 |
ALV-A | +++ | NDV | - | 鸡柔嫩艾美耳球虫 | - |
ALV-B | +++ | AIV | - | 霍乱弧菌 | - |
ALV-J | ++ | IBDV | - | ||
ALV-K | +++ | REV | - |
实施例5特异性抗体的中和活性及稳定性鉴定
在实验室采用微量细胞中和试验进行验证。将上述筛选获得的抗体用生理盐水进行倍比稀释1:2~1:128,将1份免疫血清作为阳性对照,也进行1:2~1:128的倍比稀释,每个稀释度设置5个重复。在稀释好待检样本的培养板各孔中均加入含100TCID50的病毒液,同时设置阴性血清对照的倍比稀释孔,补加等量的稀释液。将所有培养板放入37℃细胞培养箱中和2h。中和后每孔加Vero细胞悬液,放置37℃培养箱培养5d。同时设正常细胞对照。5d后观察并计数每个培养孔是否发生病变,即能够保护50%细胞不受100TCID50病毒感染的血清最高稀释度为该血清的抗体滴度,全程试验重复3次。上述抗体最终检测效价为1:64、阳性对照血清的中和效价为1:32(≧1:4判定为阳性)。中和活性试验结果表明,上述抗体具有较高的中和效价(1:64),3次重复试验的变异系数<3%,说明该抗体具有良好稳定性。为下一步抗体的鉴定提供了良好的基础。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (7)
1.一种包含禽白血病病毒多亚型抗原表位融合蛋白,所述融合蛋白中含有A、B、J、K亚群的特异性识别抗原表位,可以同时实现对常见ALV的快速筛选,其特征在于所述多亚型抗原表位融合蛋白的氨基酸序列为RKLLVSCLKLGGGSSPRSDWSIDTLSRRYCGNEFTSARGGSSDILKSRTILANSGICFQDCLSRTGGSQSREINETEPFSFMNLVLCVTGEV(SEQ ID NO.1)。
2.编码如权利要求1所述多亚型抗原表位融合蛋白的核苷酸序列。
3.一种重组表达载体,其特征在于所述重组表达载体含有权利要求2所述的DNA分子。
4.一种工程菌,其特征在于所述工程菌含有权利要求3所述的重组表达载体。
5.一种禽白血病病毒的多亚型识别抗体,其特征在于由权利要求1所述多亚型抗原表位融合蛋白免疫动物制备而成的单克隆抗体或多克隆抗体。
6.一种检测试剂盒,其特征在于所述检测试剂盒含有:
(1)权利要求1所述的禽白血病病毒多亚型抗原表位融合蛋白;
和/或
(2)权利要求5所述的多亚型识别抗体。
7.权利要求1所述多亚型抗原表位融合蛋白或权利要求5所述的多亚型识别抗体或权利要求6所述检测试剂盒的应用,所述应用为鸡种群禽白血病病毒的快速筛选和净化,所述的鸡种群禽白血病病毒的快速筛选和净化是非诊断目的以及非治疗目的的。
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