CN114957489B - 一种猪轮状病毒重组蛋白及其应用 - Google Patents
一种猪轮状病毒重组蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了一种重组猪轮状病毒蛋白,通过将猪γ干扰素以及猪轮状病毒基因的优势片段通过连接子融合后,构建成功原核表达载体,并进一步诱导制备获得了重组猪γ干扰素‑猪轮状病毒VP7蛋白。通过将纯化的重组蛋白肌肉免疫妊娠母猪,并在产后的母猪中检测到较高含量的抗PoRV IgA,证明制备重组的蛋白具有较高的免疫原性。通过对产后仔猪的攻毒实验证明,免疫重组猪γ干扰素‑猪轮状病毒VP7蛋白的母猪产下的乳猪对于轮状病毒具有更高的免疫保护作用。因此,将重组猪γ干扰素‑猪轮状病毒VP7蛋白作为制备免疫试剂,在防治母猪以及仔猪的病毒性腹泻和死亡方面具有较高的意义,值得推广应用。
Description
技术领域
本发明属于重组蛋白疫苗领域,具体涉及一种猪轮状病毒重组蛋白及该重组蛋白的应用。
背景技术
猪腹泻是一种常见的猪肠道疾病,致病机理复杂,可由细菌、病毒感染,也可以通过寄生虫等感染引起,其中猪病毒性腹泻因其感染速度快、而且范围广,危害尤为严重。引起猪病毒性腹泻的病毒种类较多,其中猪流行性腹泻病毒(Porcine epidemic diarrheavirus,PEDV)、猪轮状病毒(Porcine rotarvirus PoRV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virusof swine,TGEV)是引起猪病毒性腹泻的主要病原,其均属于RNA病毒。发病哺乳母猪腹泻,食欲减退,无乳,病仔猪表现为精神沉郁,食欲下降,排灰白色水样稀便,同时出现呕吐,严重者食欲废绝,皮肤干燥,失去弹性,极度消瘦,并脱水死亡。自2010年以来,我国猪病毒性腹泻的发病率呈明显上升的趋势。
PoRV为呼肠弧病毒科,轮状病毒属,粒子呈球状,无囊膜,有双层衣壳,直径大小为60-75nm。PoRV粒子由外层衣壳(VP4和VP7)、中层衣壳(VP6)和内衣壳(VP1、VP2和VP3)构成,中间层衣壳(VP6)有32个呈放射状排列的圆柱形壳粒组成,壳粒由此向外呈辐射状排列,外衣壳厚约20nm,为连接于壳粒末端的光滑薄膜状结构,使该病毒形成车轮状外观,轮状病毒为此而得名。PoRV由11个分节段dsRNA组成,全长约为18Kb,11个片段平均长度约为660-3300bp,经SDS-PAGE分析,每个节段含有一个顺反子、分别编码一种蛋白。基因组分别含有6个结构蛋白VP1、VP2、VP3、VP4、VP6和VP7和5个非结构蛋白Nsp1、Nsp2、Nsp3、Nsp4和Nsp5。
猪轮状病毒外衣壳蛋白主要由VP8与VP7组成,VP8与VP7是猪轮状病毒外膜上的主要中和抗原,能够刺激机体产生体液免疫和黏膜免疫,从而激发机体的免疫保护作用,因此具有以VP8与VP7为基础研制抗猪轮状病毒疫苗的潜力。
猪轮状病毒(Porcinerotavirus,PoRV)是一种常见的引起猪病毒性腹泻的病原,该病原感染后主要表现为消化道障碍,临床特征为腹泻、脱水、厌食以及体重减轻。轮状病毒可以单独感染引起腹泻、但通常与冠状病毒混合感染。有时也和大肠杆菌共同感染。轮状病毒对外界抵抗力强,病毒在18-20℃可长存7-9个月,在PH3-9的溶液中可以长期存活。PoRV可感染各种日龄的猪,感染率可高达80%-90%,1周龄仔猪尤为易感。PoRV感染发病急、传播速度快,范围广,其发病在时间上呈周期性,多发生于春天和冬天寒冷季节。
干扰素(interferon,IFN)是一类具有广谱抗病毒、抗细菌、抗肿瘤和免疫调节等多种作用的糖蛋白,具有明显的种属特异性。按照国际标准可将干扰素分为Ⅰ(IFN-α、IFN-β)、Ⅱ(IFN-γ)和Ⅲ(IFN-λ)3类,其中以α-干扰素的抗病毒作用最强。利用重组猪α-干扰素治疗猪流行性腹泻(口服,3mL/(头·次),2次/d,连用2~3d),仔猪存活率为50%~70%,但在不同猪场使用效果差异较大(存活率37.5%~70.0%不等),可能与初生仔猪感染程度不同有关。对于严重病例疗效不佳,原因可能是仅用重组猪α-干扰素不能缓解脱水和酸中毒症状,总体来说用于预防的效果,但防治成本较高。研究显示利用重组猪α-干扰素治疗猪流行性腹泻,治愈率为9
2%。此外,猪白细胞干扰素(冻干型)治疗猪流行性腹泻也有较好效果。但是,干扰素必须在病毒感染早期(此时体内病毒尚未广泛散布和引起严重病变)使用效果才佳,且需反复多次使用。
现有技术中,将干扰素γ作为免疫佐剂与猪腹泻病毒一起施用,能够有效降低猪的腹泻率和死亡率。另外,将重组干扰素对于腹泻猪进行肌肉注射也能提高猪的免疫效果。但现有技术中将干扰素与病毒外壳蛋白重组制备抗原作为猪腹泻的疫苗还鲜有报道。
考虑到干扰素以及猪轮状病毒外壳蛋白均表现出的对于病毒的免疫作用,为了获得更好的抗病毒试剂,本发明提供了一种重组的猪干扰素-轮状病毒VP7蛋白用作防治由轮状病毒引起的腹泻。
发明内容
有鉴于此,本发明的目的在于提供一种制备防治猪腹泻的重组疫苗的基因、蛋白。
为实现本发明目的,本发明提供了如下技术方案。
本发明公开了一种重组猪轮状病毒蛋白。
优选的,所述重组猪轮状病毒蛋白为重组的猪干扰素-轮状病毒蛋白。
优选的,所述重组猪轮状病毒蛋白为重组的猪干扰素-轮状病毒VP7蛋白。
优选的,所述重组猪轮状病毒蛋白为重组的猪γ干扰素-轮状病毒蛋白。
优选的,所述蛋白的序列如SEQ ID NO:4所示。
本发明公开了一种核苷酸分子,所述核苷酸分子编码所述的重组蛋白。
优选的,所述核苷酸分子的序列如SEQ ID NO:3所示。
本发明公开了一种表达载体,所述表达载体含有所述的核苷酸分子。
优选的,所述表达载体为原核表达载体。
优选的,所述表达载体为pet-28a(+)。
本发明公开了重组菌,所述重组菌包含所述的表达载体。
优选的,所述重组菌为大肠杆菌。
优选的,所述重组菌为大肠杆菌DE3。
本发明公开了扩增所述重组基因的引物对。
优选的,所述引物对包括上游引物和下游引物,所述引物对扩增所述的核苷酸分子。
优选的,所述引物对的上游引物序列如SEQ ID NO:5所示,所述引物对的下游引物序列如SEQ ID NO:6所示。
本发明公开了一种PCR反应体系,包括所述的引物对和TAQ PREMIX反应液。
本发明公开了一种PCR反应体系,包括:
2×Taq Premix 10.0μL
模板1.0μL
正义引物(25pmol/L)1.0μL
反义引物(25pmol/L)1.0μL
RNase-Free ddH2O 7.0μL
总体积20.0μL。
本发明公开了一种PCR反应的扩增条件,所述扩增条件为:95℃预变性5min;94℃变性30s,56℃复性30s,72℃延伸60s,32个循环;72℃延伸10min。
本发明公开了所述的蛋白,所述的核苷酸分子,所述的表达载体在制备猪轮状病毒免疫试剂中的应用。
本发明公开了一种猪轮状病毒免疫试剂,包括所述的蛋白和/或所述的核苷酸分子和/或所述的表达载体。
优选的,还包括佐剂。
优选的,所述佐剂选自KL02、MF59、氢氧化铝、磷酸铝、CpG佐剂。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7基因的设计合成,通过柔性连接子连接。
优选的,所述连接子为ggcggcggcggcagc(编码GGGGS连接子片段)进行连接融合。
本发明公开了通过获得的重组猪γ干扰素-猪轮状病毒VP7基因,设计扩增引物。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7蛋白的表达以及纯化。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7基因的表达载体的构建。
本发明公开了一种双酶切反应体系,包括:
胶回收产物/pET-28a(+)质粒5.0μL
Sac I 0.5μL
Xho I 0.5μL
10×Cut Buffer 1.0μL
RNase-Free ddH2O 3.0μL
总体积10.0μL。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7基因的表达方法,包括:
(1)挑选单菌落,接种到LB液体培养基中,振荡过夜。
(2)取1m L培养菌液加入到200m L LB液体培养基中,振荡培养约5h至培养基OD600=0.7左右。
(3)加入IPTG诱导表达。
(4)诱导表达结束后,收集菌体。
(5)对菌体进行超声波裂解后离心,分别收集上清和沉淀进行SDS-PAGE电泳检测。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7蛋白的纯化方法,包括:
(1)准备5mL诱导表达的重组菌。
(2)加1mL预冷的PBS充分悬浮菌体,离心弃上清,如此反复悬浮离心3次。
(3)加Binding Buffer重悬菌体,利用超声破碎仪对细菌进行破碎使细菌完全裂解。
(4)加cell Lysis Reagent,轻柔混匀。
(5)加DNase I,水平摇床上振荡25min。
(6)加Ni-Particles,温和地上下翻转多次,室温静置10min。
(7)将EP管放在碳磁力架上,待镍柱全部吸附到靠近磁力架的一边,用移液枪弃去剩余液体。
(8)加Binding Buffer,充分混匀,静置,再放入磁力架,用移液枪弃去剩余液体,如此反复2次。
(9)加Elution Buffer,充分混匀,室温静置10mi n,再放入磁力架30s,收集洗脱后的目的蛋白进行电泳检测及后续实验。
本发明公开了重组猪γ干扰素-猪轮状病毒VP7蛋白的免疫效果的分析。
优选的,包括如下步骤。
1)将纯化的重组猪γ干扰素-猪轮状病毒VP7蛋白经SDS-PAGE电泳分离后;
2)置于转膜槽中冰浴转膜,90V电压转膜;
3)使用市售的抗猪轮状病毒VP7蛋白多克隆抗体作为一抗,进行WesternBoltting杂交鉴定。
本发明通过将猪γ干扰素以及猪轮状病毒基因的优势片段通过连接子融合后,构建成功原核表达载体,并进一步诱导制备获得了重组猪γ干扰素-猪轮状病毒VP7蛋白。
通过将纯化的重组蛋白肌肉免疫妊娠母猪,并在产后的母猪中检测到较高含量的抗PoRV IgA,证明制备重组的蛋白具有较高的免疫原性。通过对产后仔猪的攻毒实验证明,免疫重组猪γ干扰素-猪轮状病毒VP7蛋白的母猪产下的乳猪对于轮状病毒具有更高的免疫保护作用。本发明的重组猪γ干扰素-猪轮状病毒VP7蛋白,通过免疫母猪后,对于产下的仔猪具有较好的轮状病毒的免疫保护效果,效果明显好于单独使用干扰素γ或VP7蛋白的免疫组。
因此,将重组猪γ干扰素-猪轮状病毒VP7蛋白作为制备免疫试剂,在防治母猪以及仔猪的病毒性腹泻和死亡方面具有较高的意义,值得推广应用。
附图说明
图1为重组质粒的PCR鉴定电泳图;
图2为重组质粒的酶切电泳图;
图3为重组菌的SDS-PAGE电泳检测结果;
图4为重组猪γ干扰素-猪轮状病毒VP7蛋白的纯化电泳图;
图5为重组猪γ干扰素-猪轮状病毒VP7蛋白的WESTERN免疫印迹分析。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1重组猪γ干扰素-猪轮状病毒VP7基因的设计合成
在NCBI上根据已经公开的猪γ干扰素基因的序列,根据软件分析选择其保守功能域结构区基因序列,获得的序列片段为:
如SEQ ID NO:1所示。
根据已经公开的不同型的猪轮状病毒的基因序列,根据软件分析获得其保守结构区以及进一步分析其优势抗原表位结构区基因序列,获得的序列片段为:
如SEQ ID NO:2所示。
将这两个基因片段之间通过柔性连接子ggcggcggcggcagc(编码GGGGS连接子片段)进行连接融合后,获得重组序列为:
如SEQ ID NO:3所示。
并交由生物公司进行重组基因的合成,获得重组猪γ干扰素-猪轮状病毒VP7基因。
该重组基因对应的编码蛋白序列为:
PVPTFKPTTTRKGCHMGQFQSLSPQELKGFKKAKDALEESLSLKNWSCSSHLFPRTRDLRQLQVWERGGGGSAPLIKAQNYGINLPITGSMDTPYMNSTTSETFLTSTLCLYYPNEAATEIADTKWTETLSQLFLTKGWPTGSVYFKGYADIASFSVEPQLYCDYNIVLMKYDGNLQLDMSELAGLILNEWLCNPMDIMLYYYQQT,如SEQ ID NO:4所示。
实施例2重组猪γ干扰素-猪轮状病毒VP7基因的表达载体的构建
根据获得的重组猪γ干扰素-猪轮状病毒VP7基因,设计扩增引物,通过对该序列进行限制性酶切位点分析后,在引物的5’端添加酶切位点,获得的扩增引物序列如下:
正义引物:5’-gagctaatgcctgtcccca ctttcaag-3’如SEQ ID NO:5所示,其中在5’端引物SacI酶切位点;
反义引物:5’-ctcgagtgtttgctgataataatat-3’如SEQ ID NO:6所示,其中在5’端引入XhoI酶切位点。
以合成的重组基因为模板,以设计的正义及反义引物为特异性引物,利用PCR方法进行重组基因的扩增,PCR体系如下:
2×Taq Premix 10.0μL
模板1.0μL
正义引物(25pmol/L)1.0μL
反义引物(25pmol/L)1.0μL
RNase-Free ddH2O 7.0μL
总体积20.0μL
反应条件:95℃预变性5min;94℃变性30s,56℃复性30s,72℃延伸60s,32个循环;72℃延伸10min。
PCR反应结束后,回收PCR产物,并用限制性内切酶分别对PCR产物以及表达载体pet-28a(+)进行酶切。限制性内切酶Sac I和Xho I胶回收PCR产物和表达载体pET-28a(+)质粒,双酶切体系如下:
胶回收产物/pET-28a(+)质粒5.0μL
Sac I 0.5μL
Xho I 0.5μL
10×Cut Buffer 1.0μL
RNase-Free ddH2O 3.0μL
总体积10.0μL。
混匀后于37℃,水浴反应2小时左右。
将酶切的产物连接后,转化大肠杆菌(DE3)后筛选阳性克隆,并提取质粒进行PCR以及酶切鉴定。
其中重组质粒的PCR鉴定电泳图参见图1,重组质粒的酶切电泳图参见图2,图1和图2的片段大小于预期符合,说明已经将重组基因构建到表达载体pET-28a(+)质粒,获得pET-28a-INFγ-VP7表达载体。进一步对构建的载体进行测序,测序结果也与设计的重组基因序列一致,表明载体构建正确。
实施例3重组猪γ干扰素-猪轮状病毒VP7基因的表达及纯化
3.1重组猪γ干扰素-猪轮状病毒VP7基因的表达
(1)从铺有pET-28a-INFγ-VP7表达载体(DE3)的平板中挑选一个单菌落,接种到5mL含50μg/m L卡那青霉素的LB液体培养基中,37℃,250r/min振荡过夜。
(2)取1m L培养菌液加入到200m L含50μg/m L卡那青霉素的LB液体培养基中,37℃,250r/min振荡培养约5h至培养基OD600=0.7左右。
(3)加入IPTG至终浓度为1mmol/L诱导表达,37℃,250r/min振荡诱导培养4h。
(4)诱导表达结束后,4℃,12,000rpm离心10min,收集菌体。
(5)对菌体进行超声波裂解后离心,分别收集上清和沉淀进行SDS-PAGE电泳检测。
电泳检测结果参见图3。其中,条带1为未诱导菌沉淀,条带3为诱导菌超声波裂解后的上清液,条带2和条带4为诱导菌超声波裂解(振幅45%,20min,开5s,避5s)后的沉淀。初步实验表明,经过IPTG诱导后,在菌体沉淀中有外源蛋白获得表达。
3.2重组猪γ干扰素-猪轮状病毒VP7蛋白的纯化(参照镍柱纯化试剂盒进行)
(1)准备5mL诱导表达的重组菌,4℃,12000rpm离心10min,弃上清,倒置2-5min。
(2)加1mL预冷的PBS充分悬浮菌体,4℃,12000rpm离心10min,弃上清,如此反复悬浮离心3次。
(3)加1mL Binding Buffer重悬菌体,利用超声破碎仪对细菌进行破碎使细菌完全裂解。
(4)加100uL的cell Lysis Reagent,轻柔混匀。
(5)加1uL的DNase I,水平摇床上振荡25min。
(6)加30uL的Ni-Particles,温和地上下翻转多次,室温静置10min。
(7)将EP管放在碳磁力架上约30s,待镍柱全部吸附到靠近磁力架的一边,用移液枪弃去剩余液体。
(8)加150uL的Binding Buffer,充分混匀,室温静置10min,再放入磁力架30s,用移液枪弃去剩余液体,如此反复2次。
(9)加100uL的Elution Buffer,充分混匀,室温静置10min,再放入磁力架30s,收集洗脱后的目的蛋白进行电泳检测及后续实验。电泳结果参见图4,表明重组蛋白能够被进一步的纯化出来。洗脱蛋白通过透析法复性以及PEG-20000浓缩后溶解于PBS中备用。
实施例4重组猪γ干扰素-猪轮状病毒VP7蛋白的免疫分析
为了进一步对纯化的蛋白进行确认,采用免疫印迹(western-BLOT)的方法对纯化的蛋白进行验证,具体操作如下。
将纯化的pET-28a-INFγ-VP7表达的蛋白经12%SDS-PAGE电泳分离后,置于转膜槽中冰浴转膜,90V电压转膜40min,使用市售的抗猪轮状病毒VP7蛋白多克隆抗体作为一抗,进行Western Boltting鉴定。结果显示在目的蛋白处出现条带(图5),说明原核表达的重组猪γ干扰素-猪轮状病毒VP7融合蛋白能够与抗猪轮状病毒VP7蛋白多克隆抗体发生免疫反应。
实施例5重组猪γ干扰素-猪轮状病毒VP7蛋白的免疫效果分析
将TGEV、PEDV抗体检测为阴性,轮状病毒(PoRV)抗原抗体双阴的8头妊娠母猪随机分成4组(A、B、C、D),每组两头。其中A组为不免疫组,另外三组(B、C、D)分别免疫重组猪γ干扰素(市售)、重组猪轮状病毒VP7蛋白(市售)以及本申请制备的重组猪γ干扰素-猪轮状病毒VP7蛋白。免疫时机为在产前30d和产前15d免疫组经肌肉注射免疫4mL(0.02mg/mL),未免疫组注射PBS4mL。
采集实验妊娠母猪于分娩后0d、3d、7d、15d乳汁样品,不少于1ml/头/次,经3000r/min,1min处理后,使用PoRV IgA ELISA抗体试剂盒对乳汁样品进行抗体测定和结果判定。
表1:A-D组不同时间段乳汁IgA抗体检测结果
编号/时间 | 0d | 3d | 7d | 15d |
A1 | 0.026 | 0.017 | 0.023 | 0.008 |
A2 | 0.014 | 0.018 | 0.009 | 0.015 |
B1 | 0.011 | 0.021 | 0.017 | 0.026 |
B2 | 0.005 | 0.019 | 0.028 | 0.014 |
C1 | 1.123 | 1.543 | 1.671 | 1.874 |
C2 | 1.213 | 1.388 | 1.532 | 1.713 |
D1 | 2.036 | 2.316 | 2.642 | 2.818 |
D2 | 2.174 | 2.514 | 2.715 | 2.901 |
从抗体检测的结果来看,A组和B组未产生相应的抗体,而C组和D组菌检测到在乳汁中有抗体形成,且D组中的抗体含量高于C组。表明本申请提供的重组蛋白具有更好的免疫原性。
实施例6攻毒保护试验
从试验组的每头母猪分娩的猪群中随机选取5头4日龄新生仔猪,分别放置于单独的盒子中,并待猪群稳定后立即口服本实验室鉴定并保存的PoRV病毒液3mL,同一窝的仔猪分别做好标记。于攻毒后6h进行饲喂,每次饲喂人工猪奶粉10-20mL。
通过攻毒后每隔6h对猪群进行观察并对各组仔猪的腹泻情况和死亡情况作详细记录,并计算出各组相应的腹泻率、死亡率。其中,攻毒后24小时的统计情况参见表2。
表2:攻毒后24h腹泻率和死亡率统计
攻毒后48小时的腹泻率和死亡率情况参见表3。
表3:攻毒后48h腹泻率和死亡率统计
从攻毒统计结果来看,本发明的重组猪γ干扰素-猪轮状病毒VP7蛋白,通过免疫母猪后,对于产下的仔猪具有较好的轮状病毒的免疫保护效果,效果明显好于单独使用干扰素γ或VP7蛋白的免疫组。
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,在不脱离本发明技术原理的前提下,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> 甘肃省畜牧兽医研究所
<120> 一种猪轮状病毒重组蛋白及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 200
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgtccccac tttcaagccc accacaacca ggaagggctg ccacatgggc cagttccaat 60
ctctgtcacc acaggagctg aagggcttca agaaagccaa ggatgctttg gaggagtcac 120
tctcactgaa gaactggagc tgcagctctc acctcttccc caggacccgg gacctgaggc 180
agctgcaggt gtgggagcgc 200
<210> 2
<211> 402
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcaccgctca ttaaagctca aaattacgga attaatttac caataactgg atctatggat 60
acgccatata tgaattcaac tacaagtgaa acatttttga cttcgacatt atgtctatat 120
tatccaaatg aagcagctac agaaattgca gatacaaaat ggacagaaac attgtcgcag 180
ttgtttttaa caaaaggatg gccaacaggg tcagtttatt ttaaaggata tgcagatatt 240
gcgtcatttt ctgtagaacc gcagttatac tgcgactata atattgtact aatgaaatat 300
gatggaaatt tacagttaga catgtctgaa ttggctggtt taatattgaa tgaatggcta 360
tgtaatccaa tggatataat gctatattat tatcagcaaa ca 402
<210> 3
<211> 618
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cctgtcccca ctttcaagcc caccacaacc aggaagggct gccacatggg ccagttccaa 60
tctctgtcac cacaggagct gaagggcttc aagaaagcca aggatgcttt ggaggagtca 120
ctctcactga agaactggag ctgcagctct cacctcttcc ccaggacccg ggacctgagg 180
cagctgcagg tgtgggagcg cggcggcggc ggcagcgcac cgctcattaa agctcaaaat 240
tacggaatta atttaccaat aactggatct atggatacgc catatatgaa ttcaactaca 300
agtgaaacat ttttgacttc gacattatgt ctatattatc caaatgaagc agctacagaa 360
attgcagata caaaatggac agaaacattg tcgcagttgt ttttaacaaa aggatggcca 420
acagggtcag tttattttaa aggatatgca gatattgcgt cattttctgt agaaccgcag 480
ttatactgcg actataatat tgtactaatg aaatatgatg gaaatttaca gttagacatg 540
tctgaattgg ctggtttaat attgaatgaa tggctatgta atccaatgga tataatgcta 600
tattattatc agcaaaca 618
<210> 4
<211> 206
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Pro Val Pro Thr Phe Lys Pro Thr Thr Thr Arg Lys Gly Cys His Met
1 5 10 15
Gly Gln Phe Gln Ser Leu Ser Pro Gln Glu Leu Lys Gly Phe Lys Lys
20 25 30
Ala Lys Asp Ala Leu Glu Glu Ser Leu Ser Leu Lys Asn Trp Ser Cys
35 40 45
Ser Ser His Leu Phe Pro Arg Thr Arg Asp Leu Arg Gln Leu Gln Val
50 55 60
Trp Glu Arg Gly Gly Gly Gly Ser Ala Pro Leu Ile Lys Ala Gln Asn
65 70 75 80
Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp Thr Pro Tyr Met
85 90 95
Asn Ser Thr Thr Ser Glu Thr Phe Leu Thr Ser Thr Leu Cys Leu Tyr
100 105 110
Tyr Pro Asn Glu Ala Ala Thr Glu Ile Ala Asp Thr Lys Trp Thr Glu
115 120 125
Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro Thr Gly Ser Val
130 135 140
Tyr Phe Lys Gly Tyr Ala Asp Ile Ala Ser Phe Ser Val Glu Pro Gln
145 150 155 160
Leu Tyr Cys Asp Tyr Asn Ile Val Leu Met Lys Tyr Asp Gly Asn Leu
165 170 175
Gln Leu Asp Met Ser Glu Leu Ala Gly Leu Ile Leu Asn Glu Trp Leu
180 185 190
Cys Asn Pro Met Asp Ile Met Leu Tyr Tyr Tyr Gln Gln Thr
195 200 205
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gagctaatgc ctgtccccac tttcaag 27
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctcgagtgtt tgctgataat aatat 25
Claims (9)
1.一种重组猪轮状病毒蛋白,其特征在于,所述蛋白的序列如SEQ ID NO:4所示。
2.一种核苷酸分子,其特征在于,所述核苷酸分子编码权利要求1所述的蛋白,所述核苷酸分子的序列如SEQ ID NO:3所示。
3.一种表达载体,其特征在于,所述表达载体含有权利要求2所述的核苷酸分子。
4.根据权利要求3所述的表达载体,其特征在于,所述表达载体为原核表达载体。
5.重组菌,其特征在于,所述重组菌包含权利要求3或4所述的表达载体。
6.根据权利要求5所述的重组菌,其特征在于,所述重组菌为大肠杆菌。
7.权利要求1所述的蛋白在制备猪轮状病毒免疫试剂中的应用。
8.一种猪轮状病毒免疫试剂,其特征在于,包括权利要求1所述的蛋白。
9.根据权利要求8所述的免疫试剂,其特征在于,还包括佐剂。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010025016A2 (en) * | 2008-08-06 | 2010-03-04 | Aeras Global Tb Vaccine Foundation | Enhancement of transgene expression from virus-based vaccine vectors using suppressors of the type 1 interferon response |
CN107353346A (zh) * | 2017-08-09 | 2017-11-17 | 芜湖英特菲尔生物制品产业研究院有限公司 | 一种由猪白蛋白与猪干扰素γ组成的融合蛋白及其制备方法和一种重组猪长效干扰素γ |
CN112625095A (zh) * | 2021-01-13 | 2021-04-09 | 武汉科前生物股份有限公司 | 一种猪轮状病毒重组蛋白以及表达该蛋白的重组腺病毒和应用 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010025016A2 (en) * | 2008-08-06 | 2010-03-04 | Aeras Global Tb Vaccine Foundation | Enhancement of transgene expression from virus-based vaccine vectors using suppressors of the type 1 interferon response |
CN107353346A (zh) * | 2017-08-09 | 2017-11-17 | 芜湖英特菲尔生物制品产业研究院有限公司 | 一种由猪白蛋白与猪干扰素γ组成的融合蛋白及其制备方法和一种重组猪长效干扰素γ |
CN112625095A (zh) * | 2021-01-13 | 2021-04-09 | 武汉科前生物股份有限公司 | 一种猪轮状病毒重组蛋白以及表达该蛋白的重组腺病毒和应用 |
Non-Patent Citations (2)
Title |
---|
Oral immunization with rotavirus VP7‑CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen‑specific IgA and IgG and passive protection in mice;YUXIAN LI ET AL.;《EXPERIMENTAL AND THERAPEUTIC MEDICINE》(第15期);4866-4874 * |
VP7-LK-IFNa-2b融合蛋白基因转化番茄的研究;张泰芳;《中国优秀硕士学位论文全文数据库农业科技辑》;第2004卷(第1期);摘要,第11页,第66页附录1 * |
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