CN108727505A - 一种免疫保护组合蛋白及其免疫疫苗 - Google Patents
一种免疫保护组合蛋白及其免疫疫苗 Download PDFInfo
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- CN108727505A CN108727505A CN201810568469.3A CN201810568469A CN108727505A CN 108727505 A CN108727505 A CN 108727505A CN 201810568469 A CN201810568469 A CN 201810568469A CN 108727505 A CN108727505 A CN 108727505A
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Abstract
本发明涉及生物技术领域，公开了一种免疫保护组合蛋白及其免疫疫苗。本发明所述免疫保护组合蛋白包括Tm16蛋白和Tm‑GST蛋白；或者为Tm16和Tm‑GST的融合蛋白。本发明将Tm16蛋白和Tm‑GST蛋白联合作为免疫原免疫动物，可对动物产生针对脑多头蚴较优的免疫保护作用，且具备协同效应，能够诱导更高的特异性抗体水平，成为脑多头蚴病的防控新措施。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种免疫保护组合蛋白及其免疫疫苗。
背景技术
脑多头蚴(Coenurus cerebralis)是多头带绦虫(Taenia multiceps)的中绦期幼虫，常寄生于山羊、绵羊、牛等偶蹄类草食动物的脑和脊髓等中枢神经系统以及皮下、肌间等部位。脑多头蚴病主要引起宿主脑及中枢神经系统的功能障碍。该病常见于非洲、东南亚等经济欠发达的发展中国家，给草食动物养殖业带来巨大的经济损失。在我国，以西北、华北、东北等广大牧区及南方农牧区最为多见。此外，人也可偶然感染该病，在欧洲、非洲、巴西、以色列和美国等国家和地区已有相关报道。脑包虫病的治疗通常包括药物治疗和手术治疗，发病后期多用药物治疗常效果不佳，而手术治疗成本较高。因此，开发新的防控技术对进一步搞好牛羊脑多头蚴病的防控具有十分重要的意义。
发明内容
有鉴于此，本发明的目的在于提供一种免疫保护组合蛋白及其免疫疫苗，使得所述组合蛋白能够对动物产生针对脑多头蚴较优的免疫保护作用，且具备协同效应，能够诱导更高的特异性抗体水平。
为了实现上述目的，本发明提供如下技术方案：
一种免疫保护组合蛋白，包括Tm16蛋白和Tm-GST蛋白；或者为Tm16和Tm-GST的融合蛋白。
针对多头带绦虫抗原基因的研究更多集中在诊断抗原的筛选上，而对于疫苗候选抗原的研究却十分有限的问题。本发明通过原核表达多头带绦虫的Tm16基因(SEQ ID NO:3所示)和Tm-GST基因(SEQ ID NO:4所示)，用获得的重组Tm16蛋白和重组TmGST蛋白对山羊进行免疫，使山羊获得极为显著的对脑多头蚴的免疫保护力，成为一种新的防控手段。
在本发明具体实施方式中，所述Tm16蛋白和Tm-GST蛋白的质量比为1:1；而当所述组合蛋白为融合蛋白时，所述融合蛋白的表达基因中Tm16蛋白表达基因和Tm-GST蛋白表达基因的拷贝量比优选为1:1。
在本发明具体实施方式中，所述Tm16蛋白和Tm-GST蛋白为带有蛋白标签的重组Tm16蛋白(rTm16)和带有蛋白标签的重组Tm-GST蛋白(rTm-GST)；在本发明具体实施方式中，所述重组Tm16蛋白序列如SEQ ID NO:1所示，所述重组Tm-GST蛋白序列如SEQ ID NO:2所示。
本发明分别以rTm16蛋白、rTm-GST蛋白以及rTm16蛋白+rTm-GST蛋白加皂素免疫山羊，于末次免疫后2周进行活虫卵感染，结果显示，Tm16组、TmGST组、Tm16+Tm-GST组山羊分别获得了50％，62.5％，87.5％的减囊率，其中Tm16+Tm-GST组山羊保护效果最好(P＝0.005)。与此同时，Tm16蛋白和Tm-GST蛋白的联合使用能诱导山羊产生更高的抗TmGST-IgG水平，表明Tm16蛋白和Tm-GST蛋白之间存在协同效应，具有预料不到的增强效果。
基于此，本发明提供了所述组合蛋白在制备脑多头蚴疫苗中的应用。作为优选，所述脑多头蚴疫苗为羊脑多头蚴疫苗，更优选为山羊脑多头蚴疫苗。
根据上述应用，本发明提供了一种脑多头蚴疫苗，包括所述组合蛋白和免疫佐剂。
作为优选，所述免疫佐剂为皂素；在本发明具体实施方式中，所述免疫佐剂为皂素QuilA(Superfos,Denmark)。
在本发明具体实施方式中，所述疫苗中，组合蛋白浓度为50μg/mL。
由以上技术方案可知，本发明将Tm16蛋白和Tm-GST蛋白联合作为免疫原免疫动物，可对动物产生针对脑多头蚴较优的免疫保护作用，且具备协同效应，能够诱导更高的特异性抗体水平，成为脑多头蚴病的防控新措施。
附图说明
图1所示为重组蛋白Tm16和TmGST纯化图；其中，M：蛋白分子质量标准；1：纯化后的rTm16蛋白；2：纯化后的rTmGST蛋白；
图2所示为表1各疫苗组山羊的血清特异性IgG OD450nm值；其中，实线表示rTm16蛋白测定的临界值，虚线表示rTmGST蛋白测定的临界值；*表示混合组中，抗rTmGST-IgG水平与抗rTm16-IgG水平呈现显著差异；V1，V2，V3分别表示第一次免疫，第二次免疫和第三次免疫。
具体实施方式
本发明公开了一种免疫保护组合蛋白及其免疫疫苗，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述组合蛋白及其免疫疫苗经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的组合蛋白及其免疫疫苗进行改动或适当变更与组合，来实现和应用本发明技术。
本发明所述蛋白标签指利用体外DNA重组技术，与Tm16蛋白和/或Tm-GST蛋白一起融合表达的一种多肽或者蛋白，以便于Tm16蛋白和/或Tm-GST蛋白的表达、检测、示踪和纯化等，如常规使用的His标签。在本发明具体实施方式中，所述重组Tm16蛋白序列为在Tm16蛋白序列5’端和3’端各有一段6个His的His标签，所述重组Tm-GST蛋白序列为在Tm-GST蛋白序列5’端和3’端各有一段6个His的His标签，用于蛋白表达分离以及纯化。
在本发明具体实施例中，所使用的Tm16蛋白和Tm-GST蛋白通过购自四川农业大学动物寄生虫病研究中心的Escherichia coli BL21-pET32a(+)-rTm16和BL21-pET32a(+)-rTmGST表达和纯化，获得带有组氨酸标签的重组蛋白rTm16和rTm-GST进行相关试验，其上所带His标签仅便于表达和纯化。
其中，表达和纯化的步骤如下：
取BL21-pET32a(+)-rTm16/BL21-pET32a(+)-rTmGST表达菌株分别接种到含50μg/mL Amp的LB培养液中，37℃培养6h直至菌液OD值达到0.6，再加入1mM IPTG诱导表达6h。随后使用NGCTM 10中高压层析系统(Bio-Rad USA)对所表达的重组蛋白进行纯化，将收集得到的蛋白进行超滤，并用SDS-PAGE鉴定。使用BCA试剂盒进行蛋白浓度测定。
E.coli BL21-pET32a(+)-rTm16和E.coli BL21-pET32a(+)-rTmGST经1mM IPTG在37℃诱导6h后，分别得到约30kDa和41kDa的可溶性蛋白，并且蛋白稳定(注：pET32a(+)的His标签约18kDa)，表明成功表达出重组Tm16蛋白和重组TmGST蛋白。对所表达的重组蛋白通过Ni-NTA亲和层析柱纯化，结果显示条带单一，纯度较好，见图1。
对rTm16蛋白、rTmGST蛋白以及rTm16+rTmGST蛋白进行冻干处理，结果获得淡黄色固体，用于试验验证免疫效果。
试验动物为44只3-4月龄攀枝花本地黑山羊(雌雄各半)，由攀枝花农林科学研究院提供。试验前1个月全部羊只免疫小反刍兽疫苗，使用伊维菌素和丙硫咪唑进行试验前驱虫处理。
试验分组见表1。
表1试验动物分组
使用无菌生理盐水稀释表1中Tm16/TmGST/Tm16+TmGST组疫苗以及皂素组，稀释后单疫苗组蛋白浓度为25μg/mL，混合组蛋白浓度为50μg/mL，注射剂量为2mL/只。
免疫程序：采用颈部皮下注射。免疫程序为首免后1个月进行二次免疫，首免后7个月进行第三次免疫。
采血：免疫前、后定期对各组山羊进行颈部静脉采血，分离血清后保存于-20℃冰箱。
人工感染:第三次免疫2周后，全部试验山羊按照约5500枚/羊，口服攻击多头带绦虫活虫卵(虫卵由四川农业大学动物寄生虫病研究中心提供)。
以下就本发明所提供的一种免疫保护组合蛋白及其免疫疫苗做进一步说明。
实施例1：Tm16+TmGST蛋白对脑多头蚴病包囊的影响
表1中TmGST疫苗组和皂素对照组在口服攻击多头带绦虫的虫卵后约1～2周，分别有1，3只羊死亡(见表2)，剖检发现脑组织有脑炎病变，同时，死前伴有体温升高、厌食、精神萎靡等脑膜脑炎症状。经口感染多头带绦虫的虫卵后105天(约3.5个月)，各组山羊全部进行剖检，对照组全部发现有包囊存在，疫苗组脑多头蚴包囊感染情况见表2。由表2可知，脑多头蚴包囊在皂素对照组中全部位于山羊的脑和脊髓部，平均直径约3cm，而联合免疫组包囊全部位于肌肉组织间，平均直径约1.5cm。相较于Tm16+TmGST联合疫苗组，Tm16疫苗组和TmGST疫苗组山羊在脑、脊髓和肌间组织部位均有包囊分布，其中有46％(6/13)的包囊位于肌间组织。
通过计算减囊率，减囊率公式如下：
应用The Mann–Whitney U test进行各疫苗组相对于对照组的保护效果评价。同时使用SPSS 20.对各试验组感染脑多头蚴包囊数量进行统计分析。
同时注射Tm16+TmGST联合疫苗的山羊获得最大的减囊率(87.5％)，而注射Tm16疫苗、TmGST疫苗的山羊分别获得50％、62.5％的减囊率。与对照组相比，Tm16与TmGST的联合免疫显著地提高了试验山羊对多头带绦虫虫卵的抵抗能力(P＝0.005)，而单独使用Tm16或TmGST则差异不显著(P＝0.172；P＝0.06)。
表2各组山羊感染脑多头蚴包囊数量情况
注：a The Mann–Whitney U test被用于检验接种山羊相对于对照组山羊包囊数量的差异性，P＜0.05表示差异显著，P＜0.01表示差异极显著；
b黑色加粗加下划线数字表示攻虫以后死亡的山羊；
实施例2：Tm16+TmGST蛋白的血清抗体的间接ELISA检测
采用间接ELISA方法，使用rTm16、rTmGST分别作为免疫抗原对各免疫组山羊血清进行特异性IgG抗体检测(联合免疫组使用rTm16和rTmGST分别进行检测)。使用SPSS 20.对各试验组特异性抗体OD450nm值进行统计分析。
使用rTm16与rTmGST蛋白分别作为检测抗原，采用间接ELISA方法进行血清特异性IgG的检测，表1各组血清IgG的OD450nm值变化见图2。从图2可以看出，首免后，TmGST组，Tm16组，联合免疫组山羊血清IgG值有明显上升，进行二次免疫后各组山羊IgG达到最高，随后缓慢下降，但仍高于临界值。二免后半年进行三免，各免疫组特异性IgG迅速上升，其中TmGST疫苗组上升最明显。
整个试验期间，除去32周所检测值外，由rTmGST所诱导的TmGST疫苗组特异性IgG水平均低于由rTm16诱导的Tm16疫苗组的IgG水平且在攻虫前具有显著差异(P＜0.05)；联合免疫组中Tm16抗体水平高于TmGST的抗体水平，仅在第4、12、14周差异显著(P＝0.14；P＝0.01；P＝0.08)随着试验的推进，Tm16疫苗组山羊的特异性IgG变化情况相比TmGST疫苗组更大。同时，在虫卵攻击以前，注射TmGST疫苗所诱导的血清抗rTmGST-IgG水平均低于联合免疫(rTm16和rTmGST蛋白)时所诱导的抗rTmGST-IgG水平，普遍呈现显著差异(P＜0.05)，攻虫以后，两组山羊的抗rTmGST-IgG水平差异不明显(P＞0.05)；而由rTm16蛋白所诱导的抗rTm16-IgG水平在单疫苗组和联合免疫组之间差异不明显(P＞0.05)，这些结果表明，rTm16与rTmGST联合使用时对山羊抗rTmGST-IgG的诱导产生了协同作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 四川农业大学
<120> 一种免疫保护组合蛋白及其免疫疫苗
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Claims (10)
1.一种免疫保护组合蛋白，其特征在于，包括Tm16蛋白和Tm-GST蛋白；或者为Tm16和Tm-GST的融合蛋白。
2.根据权利要求1所述组合蛋白，其特征在于，所述Tm16蛋白和Tm-GST蛋白的质量比为1:1。
3.根据权利要求1所述组合蛋白，其特征在于，所述融合蛋白的表达基因中Tm16蛋白表达基因和Tm-GST蛋白表达基因的拷贝量比为1:1。
4.根据权利要求1-3任意一项所述组合蛋白，其特征在于，所述Tm16蛋白为带有蛋白标签的重组Tm16蛋白。
5.根据权利要求1-3任意一项所述组合蛋白，其特征在于，所述Tm-GST蛋白为带有蛋白标签的重组Tm-GST蛋白。
6.权利要求1-5任意一项所述组合蛋白在制备脑多头蚴疫苗中的应用。
7.根据权利要求6所述应用，其特征在于，所述脑多头蚴疫苗为羊脑多头蚴疫苗。
8.一种脑多头蚴疫苗，其特征在于，包括权利要求1-5任意一项所述组合蛋白和免疫佐剂。
9.根据权利要求8所述疫苗，其特征在于，所述免疫佐剂为皂素。
10.根据权利要求8所述疫苗，其特征在于，权利要求1-5任意一项所述组合蛋白浓度为50μg/mL。
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| CN110655564A (zh) * | 2019-10-29 | 2020-01-07 | 四川农业大学 | 一种组合蛋白及其应用 |
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