CN107245105A - HN‑VP233‑221aa融合蛋白及其制备方法和应用 - Google Patents
HN‑VP233‑221aa融合蛋白及其制备方法和应用 Download PDFInfo
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- CN107245105A CN107245105A CN201710513388.9A CN201710513388A CN107245105A CN 107245105 A CN107245105 A CN 107245105A CN 201710513388 A CN201710513388 A CN 201710513388A CN 107245105 A CN107245105 A CN 107245105A
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Abstract
本发明公开一种HN‑VP233‑221aa融合蛋白及其制备方法和应用，包括鸡传染性法氏囊病毒VP233‑221aa的氨基酸序列、新城疫病毒血凝素‑神经氨酸酶（HN蛋白）的氨基酸序列以及位于VP233‑221aa氨基酸序列和新城疫病毒HN 蛋白的氨基酸序列之间的柔性Linker肽氨基酸序列；由VP2主要保护性基因片段和鸡新城疫病毒HN基因通过柔性Linker肽（‑ G‑S‑）串联连接而成。本发明的重组HN‑VP233‑221aa融合蛋白能有效诱导机体产生体液和细胞免疫应答，能诱导免疫鸡产生高水平的HN特异性抗体和VP2抗体，并可促进雏鸡T淋巴细胞的增殖活化能力，对鸡传染性法氏囊强毒株和新城疫强毒株的攻击均具有很好的保护作用，从而达到“一针防两病”的效果。
Description
技术领域
本发明涉及生物制药工业中基因工程生产疫苗和免疫佐剂领域，具体涉及一种HN-VP233-221aa融合蛋白及其制备方法和应用。
背景技术
鸡新城疫（Newcastle disease，ND）作为OIE A类疾病，鸡传染性法氏囊病（Infectious bursal disease，IBD）作为B类传染病，给全球养禽业造成巨大的经济损失。在我国，鸡新城疫与传染性法氏囊病一直是严重危害养禽业发展的重要传染病。鸡新城疫传染性强、死亡率高，且感染宿主范围越来越广。2009年瑞典、荷兰、秘鲁和比利时等发生多起新城疫疫情，造成了巨大的经济损失。近年来国内仍有NDV强毒感染鸡或鹌鹑、鸽和鸵鸟的报道，而且可以从水禽肠道中分离到NDV。IBD主要侵害3-6周龄雏鸡和青年鸡，能够破坏鸡的体液免疫中枢器官—法氏囊，导致淋巴细胞功能受损，引起免疫抑制，从而影响新城疫、禽流感等疫苗的免疫效果，且对其他疾病的易感性增加。当前由于NDV及IBDV变异株、超强毒株和新毒株的流行，导致传统疫苗免疫效果有所降低，因此，预防和控制这两种烈性传染病对养禽业的发展具有重要意义，研发防控ND和IBD的新型疫苗显得尤为重要。
ND和IBD分别是由NDV和IBDV引起的禽类发病率高、死亡率高的两种病毒性疾病。NDV的血凝素-神经氨酸酶（Hemagglutinin-neuraminidase，HN）基因编码的HN蛋白在NDV致病过程中发挥着重要作用，是NDV除F糖蛋白之外另一种较大的糖蛋白，也是病毒的主要宿主保护性抗原，可诱导机体产生中和性抗体，在机体抗感染免疫中起着重要作用。VP2作为IBDV的主要结构蛋白，还是病毒衣壳的主要成分，参与病毒中和抗体的诱导、抗原及毒力的变异，是防控鸡传染性法氏囊病的基因工程疫苗研发的潜在靶基因。
发明内容
本发明的目的是提供一种HN-VP233-221aa融合蛋白及其制备方法和应用，该融合蛋白能诱导雏鸡机体产生高水平的HN特异性抗体和VP2抗体，对鸡传染性法氏囊强毒株和新城疫强毒攻击均具有很好的保护作用。
本发明所采用的技术方案是：HN-VP233-221aa融合蛋白，包括传染性法氏囊病毒的VP2主要保护性抗原的氨基酸序列、新城疫病毒的血凝素-神经氨酸酶HN的氨基酸序列以及柔性Linker肽氨基酸序列。
作为本发明一种HN-VP233-221aa融合蛋白的进一步优化，所述HN-VP233-221aa融合蛋白由鸡传染性法氏囊病毒VP2主要保护性抗原的氨基酸序列和鸡新城疫病毒HN的氨基酸序列通过柔性Linker肽串联连接而成。
作为本发明一种HN-VP233-221aa融合蛋白的进一步优化，所述融合蛋白具有SEQ IDNO.7所示的氨基酸序列；所述的柔性Linker肽具有SEQ ID NO.6所示的氨基酸序列。
所述的具有SEQ ID NO.7所示的氨基酸序列通过一种分离的DNA序列进行编码，该DNA序列具有SEQ ID NO.5所示的核苷酸序列。
所述DNA序列的载体为原核表达载体pET32a。
所述DNA序列的宿主细胞为Rosetta大肠杆菌。
一种重组HN-VP233-221aa融合蛋白的制备方法，包括以下步骤：
（a）获得编码HN-VP233-221aa融合蛋白的基因序列；
（b）将步骤（a）获得的HN-VP233-221aa融合蛋白的基因序列，插入到原核表达载体pET32a中，得到重组载体pET32a-HN-VP233-221aa；
（c）将步骤（b）获得的重组载体pET32a-HN-VP233-221aa，转化至大肠杆菌Rosetta细胞中，经抗性筛选和PCR鉴定，获得表达融合蛋白的基因重组大肠杆菌菌株；
（d）将步骤（c）获得的表达融合蛋白的基因重组大肠杆菌菌株，在适宜的培养条件下培养，分离培养液上清，获得融合蛋白。
本发明所述融合蛋白在鸡新城疫和传染性法氏囊病的预防和治疗中的应用，该融合蛋白能诱导免疫鸡产生高水平的HN特异性抗体和VP2抗体，对鸡传染性法氏囊强毒株和新城疫强毒攻击均具有很好的保护作用。
与现有技术相比，本发明至少具有下述优点及有益效果：
1、本发明的重组HN-VP233-221aa融合蛋白能有效增强机体的体液和细胞免疫应答，能诱导免疫鸡产生高水平的HN特异性抗体和VP2抗体，对鸡传染性法氏囊强毒株和新城疫强毒攻击均具有很好的保护作用，从而达到“一针防两病”的效果。新城疫病毒强毒攻毒试验表明HN-VP233-221aa融合蛋白可使实验鸡保护率达88.9%，具有良好的免疫保护率。传染性法氏囊病毒标准毒攻毒实验表明HN-VP233-221aa融合蛋白对试验鸡的免疫保护率可达到83.3%，对防控传染性法氏囊病有很好的保护作用。
2、本发明构建的融合蛋白HN-VP233-221aa由VP2主要保护性基因片段VP233-221aa和NDV HN 基因通过柔性Linker肽（- G-S-）串联连接而成，选取抗原肽构建融合蛋白，既有助于融合蛋白以上清形式表达，又提高了融合蛋白的抗原性。
3、本发明重组的HN-VP233-221aa融合蛋白在生产时选择大肠杆菌表达系统，与传统灭活疫苗及减毒活疫苗相比，技术简单，成本低，产量高，能大规模发酵，易于实现大量生产。
4、本发明提供的HN-VP233-221aa融合蛋白，免疫鸡后不仅能产生较高水平的特异性抗体，而且对强毒攻击有很好的保护作用，并且本发明为制备重组HN-VP233-221aa融合蛋白提供了切实可行的技术路线，按本方法制备的HN-VP233-221aa融合蛋白可作为防控新城疫及传染性法氏囊病的新型基因工程疫苗，具有广阔的应用前景。
附图说明
图1为重组质粒pET32a-HN-VP233-221aa的PCR鉴定，
图中M表示5000bp DNA marker，1-2表示筛选阳性菌的PCR鉴定结果；
图2为重组质粒pET32a-HN-VP233-221aa的双酶切鉴定，
图中M表示5000bp DNA marker，1表示筛选阳性菌的PCR鉴定结果；
图3为HN-VP2融合蛋白的SDS-PAGE分析，
图中M表示蛋白分子质量标准，1表示 pET32a-HN-VP233-221aa诱导，2表示空载体对照；
图4为Western blotting 结果，
图中M表示蛋白分子量标准，1表示pET32a-HN-VP233-221aa诱导，2表示空载体对照；
图5为IBDV-VP2抗体水平分析；
图6为NDV-HN抗体水平分析；
图7为免疫重组蛋白后的淋巴细胞增殖情况(HN蛋白刺激)；
图8为免疫重组蛋白后的淋巴细胞增殖情况（VP2蛋白刺激）；
图9为不同免疫组的特异性细胞因子IFN-γ检测结果；
图10为不同免疫组的特异性细胞因子IL-4检测结果。
具体实施方式
本发明提供了一种HN-VP233-221aa融合蛋白，包括鸡传染性法氏囊病毒VP233-221aa的氨基酸序列、新城疫病毒血凝素-神经氨酸酶（HN蛋白）的氨基酸序列以及位于VP233-221aa氨基酸序列和新城疫病毒HN 蛋白的氨基酸序列之间的柔性Linker肽氨基酸序列；由VP2主要保护性基因片段和鸡新城疫病毒HN基因通过柔性Linker肽（- G-S-）串联连接而成。
该蛋白具有SEQ ID NO.7所示的氨基酸序列。
所述的柔性Linker肽具有SEQ ID NO.6所示的氨基酸序列。
同时，本发明的技术方案还采用了一种分离的DNA序列，编码具有序列为SEQ IDNO.7所示的氨基酸序列的融合蛋白，该DNA序列具有SEQ ID NO.5所示的核苷酸序列。
本发明的技术方案还采用了一种原核表达载体和大肠杆菌宿主细胞，它含有具有SEQ ID NO.5所示的DNA序列。
本发明重组的融合蛋白在鸡传染性法氏囊病和新城疫的预防和治疗中的应用。
具体地：本发明的HN-VP233-221aa融合蛋白及其DNA序列如下：
HN-VP233-221aa融合蛋白基因是由鸡传染性法氏囊病毒VP2主要保护性抗原片段（97-663bp）和鸡新城疫病毒HN基因通过柔性Linker基因串联连接而成，该串联DNA序列所编码的氨基酸序列即为HN-VP233-221aa融合蛋白。该融合蛋白能诱导雏鸡机体产生高水平的HN特异性抗体和VP2抗体，对鸡传染性法氏囊强毒株和新城疫强毒攻击均具有很好的保护作用，从而达到“一针防两病”的效果。表明二者通过柔性氨基酸肽连接，在保持独立的空间结构的同时能有效发挥各自的生物学功能。
本发明还采用了一种能高效表达HN-VP233-221aa融合蛋白的基因工程菌的构建方法：
为生产成本低的HN-VP233-221aa融合蛋白，须构建一种能生产HN-VP233-221aa融合蛋白的基因工程菌。本发明选用的基因工程菌大肠杆菌Rosetta菌株，利用高表达系统pET32a，将HN-VP233-221aa融合基因连接至pET32a，重组质粒导入至Rosetta菌株。获得高表达HN-VP233-221aa融合蛋白的基因重组大肠杆菌菌株。
本发明还采用了一种可溶性的重组HN-VP233-221aa融合蛋白的制备方法：
选用大肠杆菌表达系统，将重组菌株接种在LB培养基中，在IPTG诱导下能高效表达HN-VP233-221aa融合蛋白；收集培养液上清经his-tag蛋白纯化柱纯化后，收集穿透峰，冷冻干燥，可得到纯度极高的重组HN-VP233-221aa融合蛋白。
本发明将重组HN-VP233-221aa融合蛋白免疫鸡，通过对免疫鸡进行的NDV-HN、IBDV-VP2抗体、淋巴细胞增殖、血清中IL-4 和IFN-γ的测定以及动物攻毒保护实验，评价重组HN-VP233-221aa的免疫特性。
本发明的重组HN-VP233-221aa融合蛋白的制备方法，其生产包括以下步骤：
（a）HN-VP233-221aa和HN基因序列的获得
①IBDV VP233-221aa基因序列的获得：
根据GenBank中IBDV VP2核苷酸序列(No:AF508177)，设计1对引物扩增VP233-221aa基因片段。
以前期构建的pMD18-T-VP2为模板，PCR 扩增IBDV VP233-221aa片段。扩增产物经1%琼脂糖凝胶电泳鉴定后，切下目的条带，利用普通琼脂糖凝胶DNA回收试剂盒回收纯化，其纯化产物即为VP233-221aa基因片段，并送大连宝生物工程有限公司测序；
②NDV HN基因的获得
根据GenBank中NDV HN基因的核苷酸序列（No:JF343538），设计1对引物扩增新城疫病毒HN基因片段。
将NDV 毒株通过尿囊腔途径接种9～10 日龄SPF鸡胚，收集鸡胚的尿囊液，-20℃保存待用。按照大连宝生物工程有限公司RNA 提取试剂盒说明进行NDV RNA模板的提取。并以总RNA为模板进行RT-PCR。PCR产物经1%琼脂糖凝胶电泳鉴定后，切下目的条带，利用DNA回收试剂盒回收纯化目的基因，纯化产物为新城疫病毒HN基因，并送大连宝生物工程有限公司测序；
（b）HN-VP233-221aa融合基因序列的获得
以（a）中所述①②获得的VP233-221aa基因片段和新城疫病毒HN基因为模板，应用重叠延伸PCR方法扩增HN-VP233-221aa融合基因。
PCR产物经含有溴化乙锭1%琼脂糖凝胶电泳鉴定后，切下目的条带，随后按胶回收试剂盒使用说明进行回收HN-VP233-221aa融合基因片段，并送大连宝生物工程有限公司测序。
（c）高效表达HN-VP233-221aa融合蛋白的基因工程菌株的构建
将上述获得的HN-VP233-221aa融合蛋白基因片段，插入到原核表达载体pET32a，获得重组载体pET32a-HN-VP233-221aa；然后将重组质粒转化至Rosetta大肠杆菌中，通过抗性筛选，对疑似菌落进行PCR扩增及酶切鉴定，获得表达重组融合蛋白的Rosetta（pET32a-HN-VP233-221aa）基因工程菌株；
（d）可溶性HN-VP233-221aa融合蛋白的获得
将鉴定正确的Rosetta（pET32a-HN-VP233-221aa）的单菌落接种于LB液体培养基中，37℃过夜培养，将培养好的菌液以1：100的比例转接于LB液体培养基中，37 ℃培养至云雾状，将IPTG以1：50的比例加入，诱导培养6 h，5000r/min离心10 min收集培养液上清，即为获得的HN-VP233-221aa融合蛋白。
为使本发明的内容更明显易懂，以下结合具体实施例，对本发明进行详细描述。
实施例1
一、HN-VP233-221aa融合蛋白编码基因序列的获得
（1）新城疫病毒HN基因的获得
根据GenBank中新城疫病毒HN基因的核苷酸序列（No:JF343538），利用DNA Star7.0、Primer premier6.0软件设计1对特异性引物扩增新城疫病毒HN基因片段。
P1、P2扩增新城疫病毒HN基因，引物P1 5’端引入BamHI内切酶位点，P2 5’端引入柔性接头。引物序列如下：
表1 PCR扩增所用的引物序列
将冻存的NDV 毒株经37℃水浴融化后通过尿囊腔途径接种9～10日龄非免疫鸡胚( 每个胚0.2 mL)，37℃孵化。无菌收集24～48 h 死亡鸡胚的尿囊液(有红细胞或卵黄使尿囊液变浑浊的鸡胚液弃去)，经反复冻融3次后、8 000r/min 4℃离心15min ，取上清液，-20℃保存待用。病毒RNA 的提取按照RNA 提取试剂盒说明进行操作。以提取的总RNA 4 μL为模板，加入MgCl2 3 μL，10 ×反转录buffer 2.5 μL，dNTP Mixture (10 mmol/L)2.5 μL，RNaseInhititor 0.5 μL(20U)，RT 酶0.5μL，Taq酶0.5 μL，引物P1 1 μL，引物P2 1 μL，灭菌dH2O9.5 μL,总体系25 μL。按以下反应条件进行RT-PCR反应:50℃40 s，94℃ 2 min后，以94℃45 s，53℃90 s，72℃100 s，循环30次，最后72℃延伸10 min 。PCR产物经含有溴化乙锭1%琼脂糖凝胶电泳鉴定后，切下目的条带，随后按胶回收试剂盒使用说明进行回收即为新城疫病毒HN基因。
（2）VP233-221aa基因序列的获得
根据GenBank中IBDV VP2核苷酸序列(No:AF508177)，利用DNA Star7.0、Primerpremier6.0软件设计1对特异性引物P3、P4扩增VP233-221aa。引物由大连宝生物工程公司合成。引物P3 5’端引入柔性接头，P4 5’端HindⅢ内切酶位点。引物序列如下：
表2 PCR扩增所用的引物序列
以前期构建的pMD18-T-VP2为模板，PCR扩增鸡VP233-221aa。反应体系中分别加入10×PCR Buffer，5 μL，MgCl2，3 μL；dNTP，2.5 mmol/L，4 μL；pMD18-T-VP2模板0.5 μL， 引物P3和引物P4，终浓度为10 pmol/L各2 μL；TaKaRa ExTaq 0.5 μL；灭菌超纯水，33 μL；反应条件为：95 ℃ 预变性5 min；95 ℃变性30 s；63 ℃ 退火30 s；72 ℃延伸40 s，30个循环；72℃延伸10 min。待PCR反应结束后进行琼脂糖凝胶电泳，并观察结果。PCR扩增产物经1%琼脂糖凝胶电泳鉴定后，切下目的条带，利用大连TaKaRa公司的普通琼脂糖凝胶DNA回收试剂盒回收纯化，其纯化产物即为VP233-221aa基因片段，并送大连宝生物测序。
（3）HN-VP233-221aa融合蛋白编码基因序列的获得
以上述回收纯化的HN/VP2基因片段为模板，使用P1/P4引物，经SOEing PCR技术获取HN-VP233-221aa融合片段。
PCR反应体系50 μL:10×PCR Buffer，5 μL，MgCl2，3 μL ；dNTP，10 mmol/L，1 μL；VP233-221aa和新城疫病毒HN基因作为模板各加入5 μL， 引物P1和引物P4，终浓度为20 pmol/L各2 μL；TaKaRa ExTaq 0.5 μL；灭菌超纯水，34.5 μL；
PCR反应条件：95 ℃预变性5 min；95 ℃变性30 s；63 ℃退火30 s；72 ℃延伸2 min20s，30个循环；72 ℃延伸10 min， PCR产物经含有溴化乙锭的1%琼脂糖凝胶电泳鉴定后，切下目的条带，随后按胶回收试剂盒使用说明进行回收即为HN-VP2融合蛋白编码基因（SEQ.ID.NO.5）。
二、重组质粒pET32a-HN-VP233-221aa的构建及鉴定
首先将HN-VP233-221aa连接至pMD19-T载体，然后用BamHⅠ、HindⅢ对pMD19-T-HN-VP233-221aa进行双酶切，回收融合基因片段HN-VP233-221aa。同时将pET32a用BamHⅠ、HindⅢ进行双酶切，回收载体片段。将回收后的HN-VP233-221aa与pET32a进行连接，连接体系10 µL：目的片段6 µL，载体2 µL，T4 DNA连接酶1 µL，Buffer 1 µL。连接条件12-16℃过夜。将Rosetta感受态细胞与连接产物混匀于试管内冰浴30 min，42℃热激90 s，冰浴2 min，向试管内加入400 µL LB培养基，在37℃恒温摇床内培养1.5 h，取100 µL进行凃板(LB+AMP)，37℃培养10-12 h，挑取单菌落进行 PCR 和双酶切鉴定，如图1-2，以酶切出现约5900bp载体条带和2300bp的DNA片段的质粒为阳性质粒，并将阳性质粒命名为pET32a-HN-VP233-221aa，送至上海Invitrongen公司测序。
三、表达HN-VP233-221aa融合蛋白的基因工程菌株的构建：
取上述得到的阳性重组表达载体pET32a-HN-VP233-221aa 5 μg与80μLRosetta感受态细胞混合，混匀于试管内并冰浴30 min，然后42℃热激90 s，再冰浴2 min，最后在试管中加入400 µL LB培养基，培养1.5 h，然后用100 µL进行凃板(LB+AMP)，培养10~12 h，挑单菌落进行PCR鉴定，反应体系同上，PCR反应条件：95℃预变性5 min；95℃变性30 s；63℃退火30 s；72℃延伸2 min20 s，30个循环；72℃延伸10 min，扩增出大小为2300bp基因片段，并将鉴定正确的单菌落转接LB+AMP液体培养基，提取阳性质粒，然后用BamHⅠ，HindⅢ双酶切鉴定，酶切体系为10×H buffer 1 µL，BamHⅠ 0.4 µL，HindⅢ 0.4 µL，重组质粒 8.2 µL；酶切条件均为37ºC恒温水浴锅内反应3h。PCR及酶切鉴定正确的菌落为表达HN-VP233-221aa融合蛋白的基因工程大肠杆菌菌株。
四、重组HN-VP233-221aa融合蛋白的诱导表达及鉴定：
将鉴定正确的Rosetta(pET32a-HN-VP233-221aa)单菌落转接到LB+AMP液体培养基中培养10~12h，在将培养好的菌液与培养基1:100扩大培养5 h左右。加入80 µL诱导剂IPTG（诱导剂与菌液比例为1:50），震摇6 h。之后将菌液取出，把诱导过的菌装入1.5 mL EP管中，离心，去上清，加入PBS或者超纯水200 µL洗2~3遍，最后加入200 µL超纯水用超声细胞破碎仪进行破碎，至溶液透光。之后在溶液中加入50 µL 5×的上样液（上样液与溶液比例为1:4），沸水中煮10 min，做好标记即可。
配制5 mL分离胶，用5 mL移液枪取适量加入胶板中，加入适量异丁醇消除气泡，静置约1 h待分离胶凝固；用滤纸将异丁醇完全吸出，配制2 mL浓缩胶加入胶板中，迅速插上梳子，静置1 h待胶凝固；将胶板置于电泳槽内适当位置，加入适当缓冲液，拔出梳子进行点样，连接电源，浓缩胶部分恒压80 V跑45 min左右，待溴酚蓝跑过浓缩胶，之后恒压120 V，至样品有部分溴酚蓝跑出缓冲液，断开电源。
取出电泳凝胶，用考马斯亮蓝染色1 h，随后用脱色液进行脱色，大约每2 h换1次，重复4次左右即可。在凝胶成像仪中分析蛋白表达情况。重组的HN-VP233-221aa融合蛋白分子量约104 kDa，如图3。
同时用NDV阳性血清作为一抗进行Western blotting试验，重组大肠杆菌的培养液上样的泳道有一条特异性目的条带，而阴性对照的上样泳道未出现条带，如图4。表明重组HN-VP233-221aa融合蛋白得到了成功表达，且具有很好的免疫反应性。
实施例2
重组HN-VP233-221aa融合蛋白的应用：
1）动物免疫
用上述重组HN-VP233-221aa融合蛋白免疫鸡，评价其免疫特性。将14日龄健康雏鸡240只随机分成4组，60只/组。第1组为阴性对照，免疫200 μL PBS；第2组免疫200 μg的HN-VP2重组融合蛋白（用200 μL PBS稀释），采用胸部肌肉注射方式进行免疫。第3组、第4组分别免疫鸡新城疫IV系疫苗及法氏囊疫苗（B87株）作为传统疫苗对照组，采用滴鼻、点眼的途径免疫200 μL；所有实验组均间隔2周免疫1次，共免疫3次。并分别于首次免疫接种后第7d、21d、35d时各组随机抽取8只雏鸡进行心脏采血，分离血清。同时，无菌采取脾脏进行淋巴细胞增殖试验用。于最后1次免疫后2周时，取各组试验鸡18只进行NDV F48E9标准强毒的攻击（1000 ELD50/只），剩余18只进行IBD标准毒的攻毒；
攻毒后连续观察7天，记录各组试验鸡的发病和死亡情况。
2）NDV HN抗体和IBDV VP2抗体的测定
分别于首免后7、21、35d时分离血清，通过ELISA检测每组实验鸡免疫后HN抗体和VP2IgG抗体的产生，发现重组融合蛋白HN-VP233-221aa免疫组在首免1周后就能检测到NDV-HN、IBDV-VP2抗体，在第2次加强免疫后抗体产生不断增加，抗体水平分别与传统疫苗组相当，无显著性差异（图5-6）。
3）四甲基偶氮唑蓝法（MTT）分析
从第1次免疫后第7、21、35d时无菌采取各试验鸡的脾脏，制备单细胞悬液，检测脾脏T淋巴细胞的增殖情况。结果显示免疫后不同检测点HN-VP233-221aa融合蛋白免疫组诱导T淋巴细胞增殖能力最强，在同一时期与其它各组之间均具有显著性差异（p<0.05）（图7-8）。
4）血清中IL-4 和IFN-γ 的测定
应用定量ELISA对血清中的IL-4和IFN-γ进行定量分析发现，HN-VP233-221aa融合蛋白免疫组诱导淋巴细胞分泌特异性IL-4和IFN-γ的水平最强，差异显著（p<0.05）。其次是鸡新城疫IV系疫苗免疫组和传染性法氏囊病疫苗免疫组（p<0.05）（图9-10）。
5）攻毒试验结果
在最后1次免疫接种后2周时，对各免疫组剩余18只鸡分别应用F48E9强毒攻击和IBD标准强毒攻击。结果如表3-4所示，NDV强毒攻毒情况：HN-VP2免疫组中18只试验鸡在7天观察期内有2只发病，其中1只死亡，保护率为88.9%；而IV系疫苗免疫组发病1只，保护率为94.4%。IBDV标准强毒攻毒情况：HN-VP233-221aa融合蛋白免疫组18只试验鸡在7天观察期内有3只发病，保护率达83.3%，传统疫苗组的保护率为88.9%（表1）。
表3 NDV攻毒后各组的保护率
表4 IBDV攻毒后各组的保护率
以上实施例仅用以说明，而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明。本领域的普通技术人员应当理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，其均应涵盖在本发明的权利要求范围当中。
SEQUENCE LISTING
<110> 河南科技大学
<120> HN-VP233-221aa融合蛋白及其制备方法和应用
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agtgggtggg gggcacctat ccatgaccca gattatatag gggggatagg caaagaactc 420
attgtagatg atgctagtga tatcacatca ttctatccct ctgcatttca aggacatctg 480
aattttatcc cggcgcctac tacaggatca ggttgcactc gaatacccgc atttgacatg 540
agtgctaccc attactgcta cacccataat gtaatattgt ctggatgcag agatcactca 600
cattcatatc agtatttagc acttggtgtg ctccggacat ctgcaacagg gagggtattc 660
ttttctactc tgcgttccat caacctggac gacacccaaa atcggaagtc ttgcagtgtg 720
agtgcaactc ccttgggttg tgatatgctg tgctcgaaag tcacggagac agaggaagaa 780
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taccacgaaa aggacctaga tgtcacaaca ttattcgggg actgggtggc caactaccca 900
ggagtagggg gtggatcttt tattgacagc cgcgtatggt tctcagtcta cggagggtta 960
aaacccaatt cacccagtga cactgtacag gaagggaaat atgtgatata caagcgatac 1020
aatgacacat gcccagatga gcaagactac cagattcgaa tggccaagtc ttcgtataag 1080
cctggacggt ttggtgggaa acgcatacag caggctatct tatctatcaa ggtgtcaaca 1140
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ccttatacat tcaatgcctt cactcggcca ggtagtatcc cttgtcaggc ttcagcaaga 1380
tgccccaacc cgtgtgttac tggagtctat acagatccat atcccctaat cttctataga 1440
aaccacacct tgcgaggggt attcgggaca atgcttgatg gtgtacaagc aagacttaac 1500
cctgcgtctg cagtattcga tagcacatcc cgcagtcgca ttactcgagt gagttcaagc 1560
agtaccaaag cagcatacac aacatcaact tgttttaaag tggtcaagac taataagacc 1620
tattgtctca gcattgctga aatatctaat actctcttcg gagaattcag aatcgtcccg 1680
ttactagttg agatcctcaa agatgacggg gttggatcac tggagaagca cactctcagg 1740
tcagagacct cgacctacaa tttgactgtg ggggacacag ggtcagggct aattgtcttt 1800
ttccctggat tccctggctc aattgtgggt gctcactaca cactgcagag caatgggaac 1860
tacaagttcg atcagatgct cctgactgcc cagaacctac cggccagtta caactactgc 1920
aggctagtga gtcggagtct cacagtgagg tcaagcacac ttcctggtgg cgtttatgca 1980
ctaaacggca ccataaacgc cgtgaccttc caaggaagcc tgagtgaact gacagatgtt 2040
agctacaatg ggttgatgtc tgcaacagcc aacatcaacg acaaaattgg gaacgtccta 2100
gtaggggaag gggtcaccgt cctcagctta cccacatcat atgatcttgg gtatgtgagg 2160
cttggtgacc ccattcccgc aatagggctt gacccaaaaa tggtagccac atgtgacagc 2220
agtgacaggc ccagagtcta caccataact gcagccgatg attaccaatt ctcatcacag 2280
taccaataa 2289
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Claims (9)
1.HN-VP233-221aa融合蛋白，其特征在于：包括传染性法氏囊病毒的VP2主要保护性抗原的氨基酸序列、新城疫病毒的血凝素-神经氨酸酶HN的氨基酸序列以及柔性Linker肽氨基酸序列。
2.根据权利要求1所述的HN-VP233-221aa融合蛋白，其特征在于：由鸡传染性法氏囊病毒VP2主要保护性抗原的氨基酸序列和鸡新城疫病毒HN的氨基酸序列通过柔性Linker肽串联连接而成。
3.根据权利要求1或2所述的HN-VP233-221aa融合蛋白，其特征在于：所述融合蛋白具有SEQ ID NO.7所示的氨基酸序列。
4.根据权利要求1或2所述的HN-VP233-221aa融合蛋白，其特征在于：所述的柔性Linker肽具有SEQ ID NO.6所示的氨基酸序列。
5.根据权利要求3所述的HN-VP233-221aa融合蛋白，其特征在于：所述的具有SEQ ID NO.7所示的氨基酸序列通过一种分离的DNA序列进行编码，该DNA序列具有SEQ ID NO.5所示的核苷酸序列。
6.根据权利要求5所述的HN-VP233-221aa融合蛋白，其特征在于：所述DNA序列的载体为原核表达载体pET32a。
7.根据权利要求5所述的HN-VP233-221aa融合蛋白，其特征在于：所述DNA序列的宿主细胞为Rosetta大肠杆菌。
8.一种重组HN-VP233-221aa融合蛋白的制备方法，其特征在于：包括以下步骤：
（a）获得编码HN-VP233-221aa融合蛋白的基因序列；
（b）将步骤（a）获得的HN-VP233-221aa融合蛋白的基因序列，插入到原核表达载体pET32a中，得到重组载体pET32a-HN-VP233-221aa；
（c）将步骤（b）获得的重组载体pET32a-HN-VP233-221aa，转化至大肠杆菌Rosetta细胞中，经抗性筛选和PCR鉴定，获得表达融合蛋白的基因重组大肠杆菌菌株；
（d）将步骤（c）获得的表达融合蛋白的基因重组大肠杆菌菌株，在适宜的培养条件下培养，分离培养液上清，获得融合蛋白。
9.权利要求1或2所述融合蛋白在鸡新城疫和传染性法氏囊病的预防和治疗中的应用。
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