CN111500512A - 三种表达禽流感病毒蛋白的重组乳酸菌及构建方法和应用 - Google Patents
三种表达禽流感病毒蛋白的重组乳酸菌及构建方法和应用 Download PDFInfo
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Abstract
本发明涉及三种表达禽流感病毒蛋白的重组乳酸菌及其构建方法和应用，所述表达禽流感病毒蛋白的重组乳酸菌菌株含有编码H5N6禽流感病毒NP蛋白的基因序列、编码H7N9禽流感病毒NP蛋白的基因序列或编码H9N2禽流感病毒HA蛋白的基因序列中的任意一种。所述构建方法主要为将目的基因与乳酸菌表达载体进行连接，将所述重组质粒导入ATCC393干酪乳杆菌感受态中，得到所述重组乳酸菌菌株。所述重组乳酸菌在动物体内能够表达特定蛋白，刺激机体产生具有保护作用的抗体，对预防和治疗H5、H7和H9禽流感具有重要意义。本发明还提供一种预防和治疗禽流感的口服疫苗或药物，能够有效预防和治疗H5、H7和H9禽流感。
Description
技术领域
本发明属于生物技术领域，具体涉及三种表达禽流感病毒蛋白的重组乳酸 菌及其构建方法和应用。
背景技术
禽流感病毒，属于甲型流感病毒，根据禽流感病毒对鸡和火鸡的致病性的 不同，分为高、中、低/非致病性三级。由于禽流感病毒的血凝素结构等特点， 一般感染禽类，当病毒在复制过程中发生基因重配，致使结构发生改变，获得 感染人的能力，可能造成人感染禽流感疾病的发生。
本病的临床表现有多种类型，有的呈无症状表现的隐性感染，有的呈致死 率较低呼吸道感染，有的则呈致死率很高的急性出血性感染，并可传染人。由 于其传播快，危害大，被世界动物卫生组织列为A类动物传染病，我国将其列 为一类动物疫病。禽流感的防治措施较多，其中广泛使用的为接种疫苗，由于 禽流感血清型较多，而且交叉保护性差。因此，接种疫苗时，必须针对当地流 行的亚型，选择相应的亚型疫苗免疫，方可取得良好的免疫效果。
至今发现能直接感染人的禽流感病毒亚型有：H5N1、H5N6、H7N1、H7N2、 H7N3、H7N7、H7N9和H9N2亚型。2013年3月在人体上首次发现的新禽流 感H7N9亚型。H9N2是一种禽流感的病毒，从2000年来从鸭及鸡只身上分离 出来，有研究表明H9N2病毒已经感染到了一些猪的身上。H9N2病毒在人类 身上发现比较罕见，是一种轻微的疾病，但世界卫生组织早前警告，H9N2曾 导致人类死亡，也可以是引发新一轮全球流感潮的元凶，同样需密切监察。H9 病毒在亚洲普遍存在，由于毒性较低，对人类健康的威胁较低，但需提防H9 与其他流感病毒的"洗牌"效应，制造新流感病毒的风险。H5N6、H7N9和H9N2 亚型禽流感病毒是潜在的人畜共患病，不仅造成了家禽、家畜等养殖业的重大 损失，而且可能对人类的健康造成重大威胁。
发明内容
为了解决现有技术存在的上述问题，本发明提供了表达禽流感病毒蛋白的 重组乳酸菌及其构建方法和应用。所述重组乳酸菌在动物体内能够表达特定蛋 白，刺激机体产生具有保护作用的抗体，对预防和治疗H5、H7和H9禽流感 具有重要意义。将这三种流感病毒的抗原片段，构建到乳酸菌分泌表达载体中， 转化到乳酸菌中，不需要诱导，口服或者拌料的方法就可以进行蛋白的表达， 生产和使用方便，不失为一种非常有效的方法。
本发明所采用的技术方案为：
表达禽流感病毒蛋白的重组乳酸菌菌株，含有编码H5N6禽流感病毒NP 蛋白的基因序列、编码H7N9禽流感病毒NP蛋白的基因序列或编码H9N2禽 流感病毒HA蛋白的基因序列中的任意一种。
进一步的，所述编码H5N6禽流感病毒NP蛋白的基因序列如SEQ ID NO:1 所示，所述H5N6禽流感病毒NP蛋白氨基酸序列如SEQ ID NO:2所示；所述 编码H7N9禽流感病毒NP蛋白的基因序列如SEQ ID NO:3所示，所述H7N9 禽流感病毒NP蛋白氨基酸序列如SEQ ID NO:4所示；所述编码H9N2禽流感 病毒HA蛋白的基因序列如SEQ ID NO:5所示，所述H9N2禽流感病毒HA蛋 白氨基酸序列如SEQ ID NO:6所示。
所述的表达禽流感病毒蛋白的重组乳酸菌菌株的构建方法，包括以下步骤：
(1)提取禽流感病毒RNA，进行反转录，得到cDNA；合成相应病毒基 因的扩增引物，得到引物组；
(2)用所述cDNA和所述引物组进行PCR扩增，得到扩增产物，将所述 扩增产物进行琼脂糖凝胶回收，得到目的基因；
(3)将所述目的基因与载体进行同源重组，得到同源重组产物；将所述同 源重组产物进行克隆，挑取阳性克隆进行测序验证，得到重组质粒；
(4)将所述重组质粒导入ATCC393干酪乳杆菌感受态中，之后进行涂板、 培养、挑取单克隆菌株进行鉴定，得到所述表达禽流感病毒蛋白的重组乳酸菌 菌株。
进一步的，步骤(1)中，所述病毒为H5N6禽流感病毒、H7N9禽流感病 毒或H9N2禽流感病毒中的任意一种。
进一步的，所述病毒为H5N6禽流感病毒时，合成NP基因的引物组 H5N6-NP-1077F和H5N6-NP-1077R，序列如SEQ ID NO:7和SEQ ID NO:8所 示；所述病毒为H7N9禽流感病毒时，合成NP基因的引物组H7N9-NP-1515F 和H7N9-N9-1515R，序列如SEQ ID NO:9和SEQ IDNO:10所示；所述病毒为 H9N2禽流感病毒时，合成HA基因的引物组H9N2-HA-579F和H9N2-HA-579R， 序列如SEQ ID NO:11和SEQ ID NO:12所示；所述扩增引物上游含有SalI位点，下游含EcoRV位点。
进一步的，步骤(3)中，所述载体为pVE5523载体。
进一步的，步骤(4)中，所述导入操作为：将所述重组质粒与ATCC393 干酪乳杆菌感受态轻柔混匀后，放置冰上5min，之后转入直径1mm的预冷电 转化杯中，迅速电击，电击参数为电压2.5kV，电击时间5ms；电击后加入800μL 冰预冷的MRS培养基，混匀之后转移至1.5ml离心管中，放置冰上10min，28℃ 培养4h，得到菌液；
所述涂板和培养操作为：取100μL所述菌液涂布于含有5μg/ml红霉索的 MRS固体培养基上，28℃培养3天；挑取单个菌落，分别接种于含有5ug/ml 红霉素的MRS液体培养基中，28℃培养48h。
所述表达禽流感病毒蛋白的重组乳酸菌菌株在制备预防和治疗禽流感药物 中的应用，优选的，所述药物为口服疫苗。
一种预防和治疗禽流感的药物，包含所述表达禽流感病毒蛋白的重组乳酸 菌菌株。
一种制备所述预防和治疗禽流感药物的方法，包括以下步骤：用MRS液 体培养基将所述重组乳酸菌在37℃下培养72h，离心得到菌体，将所述菌体与 辅料混匀后35℃干燥，得到粉剂；检测所述粉剂活菌数，保证活菌数>100亿/ 克，得到所述预防和治疗禽流感的药物。
进一步的，所述辅料由可溶性淀粉、脱脂乳、海藻糖、谷氨酸钠、山梨醇 和维生素C组成。
进一步的，所述辅料包括50-80％的可溶性淀粉、5-20％脱脂乳、2-10％海 藻糖、5-10％谷氨酸钠、1-5％山梨醇和1-5％维生素C。
一种预防和治疗禽流感的口服疫苗，包含所述表达禽流感病毒蛋白的重组 乳酸菌菌株经过诱导后表达的融合蛋白。
进一步的，所述融合蛋白的氨基酸序列如SEQ ID NO:2所示。
进一步的，所述融合蛋白的氨基酸序列如SEQ ID NO:4所示。
进一步的，所述融合蛋白的氨基酸序列如SEQ ID NO:6所示。
本发明的有益效果为：
本发明所述表达禽流感病毒蛋白的重组乳酸菌菌株含有编码H5N6禽流感 病毒NP蛋白的基因序列、编码H7N9禽流感病毒NP蛋白的基因序列或编码 H9N2禽流感病毒HA蛋白的基因序列中的任意一种。所述构建方法包括：提 取病毒RNA，进行反转录，得到cDNA；合成相应病毒基因的扩增引物，得到 引物组；用所述cDNA和所述引物组进行PCR扩增，得到目的基因；将目的基 因与载体进行重组，得到重组质粒；将所述重组质粒导入ATCC393干酪乳杆菌感受态中，再进行涂板、培养等，得到所述重组乳酸菌菌株。所述重组乳酸 菌在动物体内能够表达特定蛋白，刺激机体产生具有保护作用的抗体。实验证 明，本发明所述三种表达禽流感病毒蛋白的重组乳酸菌菌株能够明显提高细胞 免疫应答和体液免疫应答水平，会刺激机体产生相应种类的抗体，有效提高机 体中IgG的分泌，充分证明本发明所述菌株对预防和治疗H5、H7和H9禽流 感的重要意义。所述表达禽流感病毒蛋白的重组乳酸菌菌株在制备预防和治疗 禽流感药物中的应用，利用所述菌株制备的口服疫苗或药物能够有效预防和治 疗H5、H7和H9禽流感。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施 例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述 中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付 出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例1所述重组质粒pVE5523-H5N6-NP的图谱；
图2是本发明实施例2所述重组质粒pVE5523-H7N9-NP的图谱；
图3是本发明实施例3所述重组质粒pVE5523-H9N2-HA的图谱；
图4是本发明实施例3所述质粒pVE5523的图谱。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方 案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不 是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创 造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例1
本实施例提供表达H5N6禽流感病毒NP蛋白的重组乳酸菌的构建方法， 包括以下步骤：
(1)提取H5N6禽流感病毒RNA，进行反转录，得到cDNA；编码H5N6 禽流感病毒NP蛋白的基因序列如SEQ ID NO:1所示，合成NP基因的引物组 H5N6-NP-1077F和H5N6-NP-1077R，序列如SEQ ID NO:7和SEQ ID NO:8所 示；
(2)用所述cDNA和所述引物组进行PCR扩增，得到扩增产物，将所述 扩增产物进行琼脂糖凝胶回收，得到目的基因；
(3)将所述目的基因与pVE5523载体进行同源重组，得到同源重组产物； 将所述同源重组产物进行克隆，挑取阳性克隆进行测序验证，得到重组质粒 pVE5523-H5N6-NP，图谱如图1所示；
(4)将所述重组质粒pVE5523-H5N6-NP与ATCC393干酪乳杆菌感受态 轻柔混匀后，放置冰上5min，之后转入直径1mm的预冷电转化杯中，迅速电 击，电击参数为电压2.5kV，电击时间5ms；电击后加入800μL冰预冷的MRS 培养基，混匀之后转移至1.5ml离心管中，放置冰上10min，28℃培养4h，得 到菌液；取100μL所述菌液涂布于含有5μg/ml红霉索的MRS固体培养基上， 28℃培养3天；挑取单个菌落，分别接种于含有5ug/ml红霉素的MRS液体培 养基中，28℃培养48h，挑取单克隆菌株进行鉴定，得到所述表达H5N6禽流 感病毒NP蛋白的重组乳酸菌菌株，名称为H5N6-NP/ATCC393干酪乳杆菌。
本实施例还提供一种预防和治疗禽流感的药物，包含上述表达H5N6禽流 感病毒NP蛋白的重组乳酸菌菌株。
进一步提供一种制备所述预防和治疗禽流感的药物的方法，具体为用MRS 液体培养基将所述重组乳酸菌在37℃下培养72h，离心得到菌体，将所述菌体 与辅料混匀后35℃干燥，得到粉剂；检测所述粉剂活菌数，保证活菌数>100 亿/克，得到所述预防和治疗H5N6禽流感的药物；所述辅料包含50％的可溶性 淀粉、20％脱脂乳、10％海藻糖、10％谷氨酸钠、5％山梨醇和5％维生素C。
本实施例还提供一种预防和治疗H5N6禽流感的口服疫苗，包含上述表达 H5N6禽流感病毒NP蛋白的重组乳酸菌菌株经过诱导后表达的融合蛋白，所述 融合蛋白的氨基酸序列如SEQ ID NO:2所示。
实施例2
本实施例提供表达H7N9禽流感病毒NP蛋白的重组乳酸菌的构建方法， 包括以下步骤：
(1)提取H7N9禽流感病毒RNA，进行反转录，得到cDNA；编码H7N9 禽流感病毒NP蛋白的基因序列如SEQ ID NO:3所示，合成NP基因的引物组 H7N9-NP-1515F和H7N9-N9-1515R，序列如SEQ ID NO:9和SEQ ID NO:10所 示；
(2)用所述cDNA和所述引物组进行PCR扩增，得到扩增产物，将所述 扩增产物进行琼脂糖凝胶回收，得到目的基因；
(3)将所述目的基因与pVE5523载体进行同源重组，得到同源重组产物； 将所述同源重组产物进行克隆，挑取阳性克隆进行测序验证，得到重组质粒 pVE5523-H7N9-NP，图谱如图2所示；
(4)将所述重组质粒pVE5523-H7N9-NP与ATCC393干酪乳杆菌感受态 轻柔混匀后，放置冰上5min，之后转入直径1mm的预冷电转化杯中，迅速电 击，电击参数为电压2.5kV，电击时间5ms；电击后加入800μL冰预冷的MRS 培养基，混匀之后转移至1.5ml离心管中，放置冰上10min，28℃培养4h，得 到菌液；取100μL所述菌液涂布于含有5μg/ml红霉索的MRS固体培养基上， 28℃培养3天；挑取单个菌落，分别接种于含有5ug/ml红霉素的MRS液体培 养基中，28℃培养48h，挑取单克隆菌株进行鉴定，得到所述表达H7N9禽流 感病毒NP蛋白的重组乳酸菌菌株，名称为H7N9-NP/ATCC393干酪乳杆菌。
本实施例还提供一种预防和治疗H7N9禽流感的药物，包含上述表达H7N9 禽流感病毒NP蛋白的重组乳酸菌菌株。
进一步提供一种制备所述预防和治疗H7N9禽流感的药物的方法，具体为 用MRS液体培养基将所述重组乳酸菌在37℃下培养72h，离心得到菌体，将 所述菌体与辅料混匀后35℃干燥，得到粉剂；检测所述粉剂活菌数，保证活菌 数>100亿/克，得到所述预防和治疗H7N9禽流感的药物；所述辅料包含80％ 的可溶性淀粉、5％脱脂乳、5％海藻糖、5％谷氨酸钠、2.5％山梨醇和2.5％维生 素C。
本实施例还提供一种预防和治疗H7N9禽流感的口服疫苗，包含上述表达H7N9禽流感病毒NP蛋白的重组乳酸菌菌株经过诱导后表达的融合蛋白，所述 融合蛋白的氨基酸序列如SEQ ID NO:4所示。
实施例3
本实施例提供表达H9N2禽流感病毒HA蛋白的重组乳酸菌的构建方法， 包括以下步骤：
(1)提取H9N2禽流感病毒RNA，进行反转录，得到cDNA；编码H9N2 禽流感病毒HA蛋白的基因序列如SEQ ID NO:5所示，合成NP基因的引物组 H9N2-HA-579F和H9N2-HA-579R，序列如SEQ ID NO:11和SEQ ID NO:12所 示；
(2)用所述cDNA和所述引物组进行PCR扩增，得到扩增产物，将所述 扩增产物进行琼脂糖凝胶回收，得到目的基因；
(3)将所述目的基因与pVE5523载体(图谱如图4所示)进行同源重组， 得到同源重组产物；将所述同源重组产物进行克隆，挑取阳性克隆进行测序验 证，得到重组质粒pVE5523-H9N2-HA，图谱如图3所示；
(4)将所述重组质粒pVE5523-H9N2-HA与ATCC393干酪乳杆菌感受态 轻柔混匀后，放置冰上5min，之后转入直径1mm的预冷电转化杯中，迅速电 击，电击参数为电压2.5kV，电击时间5ms；电击后加入800μL冰预冷的MRS 培养基，混匀之后转移至1.5ml离心管中，放置冰上10min，28℃培养4h，得 到菌液；取100μL所述菌液涂布于含有5μg/ml红霉索的MRS固体培养基上， 28℃培养3天；挑取单个菌落，分别接种于含有5ug/ml红霉素的MRS液体培 养基中，28℃培养48h，挑取单克隆菌株进行鉴定，得到所述表达H9N2禽流 感病毒NP蛋白的重组乳酸菌菌株，名称为H9N2-HA/ATCC393干酪乳杆菌。
进一步提供一种制备所述预防和治疗H9N2禽流感的药物的方法，具体为 用MRS液体培养基将所述重组乳酸菌在37℃下培养72h，离心得到菌体，将 所述菌体与辅料混匀后35℃干燥，得到粉剂；检测所述粉剂活菌数，保证活菌 数>100亿/克，得到所述预防和治疗H9N2禽流感的药物；所述辅料包含65％ 的可溶性淀粉、12.5％脱脂乳、9％海藻糖、7.5％谷氨酸钠、3％山梨醇和3％维 生素C。
进一步还提供一种预防和治疗H9N2禽流感的口服疫苗，包含上述表达 H9N2禽流感病毒HA蛋白的重组乳酸菌菌株经过诱导后表达的融合蛋白，所 述融合蛋白的氨基酸序列如SEQ ID NO:6所示。
实验例
实施例1-3所得重组乳酸菌在动物体内的免疫效果检测
一、试验过程
1、实施例1所得H5N6-NP/ATCC393干酪乳杆菌饲喂试验：
选取1月龄鸡共30只，随机平均分为4组，分别为空白组、ATCC393干 酪乳杆菌组(1千克/吨饲料)、实施例1所得H5N6-NP/ATCC393干酪乳杆菌 低浓度组(0.5千克/吨饲料)和实施例1所得H5N6-NP/ATCC393干酪乳杆菌 高浓度组(1千克/吨饲料)。共饲喂2次，间隔2周。第一次饲喂后每两周取 一次血清，一共取4次血清。
2、实施例2所得H7N9-NP/ATCC393干酪乳杆菌饲喂试验：
选取1月龄鸡共30只，随机平均分为4组，分别为空白组、ATCC393干 酪乳杆菌组(1千克/吨饲料)、实施例2所得H7N9-NP/ATCC393干酪乳杆菌 低浓度组(0.5千克/吨饲料)和实施例2所得H7N9-NP/ATCC393干酪乳杆菌 高浓度组(1千克/吨饲料)。共饲喂2次，间隔2周。第一次饲喂后每两周取 一次血清，一共取4次血清。
3、实施例3所得H9N2-HA/ATCC393干酪乳杆菌饲喂试验：
选取1月龄鸡共30只，随机平均分为4组，分别为空白组、ATCC393干 酪乳杆菌组(1千克/吨饲料)、实施例3所得H9N2-HA/ATCC393干酪乳杆菌 低浓度组(0.5千克/吨饲料)和实施例3所得H9N2-HA/ATCC393干酪乳杆菌 高浓度组(1千克/吨饲料)。共饲喂2次，间隔2周。第一次饲喂后每两周取 一次血清，一共取4次血清。
4、H5N6-NP、H7N9-NP和H9N2-HA抗体水平检测：
抗体检测试剂：H5亚型禽流感(H5N6)HI试验用抗原及阳性血清；H7 亚型禽流感(H7N9)HI试验用抗原及阳性血清；H9亚型禽流感(H9N2)HI 试验用抗原及阳性血清；1％红细胞悬液：无菌操作采健康公鸡抗凝血，按常规 制备1％红细胞悬液，4℃保存备用。
参照《高致病性禽流感诊断技术》(GB/T 18936-2003)规定的微量法，采用 HI试验进行样品中H5亚型、H7亚型和H9亚型禽流感抗体检测。
判定标准：HI抗体效价≥4log2为抗体阳性；HI抗体效价＜4log2为阴性。
二、血凝和血凝抑制试验检测抗体效价结果
(1)H5抗体效价(log2)
表1-H5抗体效价(log2)
以上表1结果表明：和ATCC393干酪乳杆菌组(1千克/吨饲料)相比， H5N6-NP/ATCC393干酪乳杆菌低浓度组(0.5千克/吨饲料)和 H5N6-NP/ATCC393干酪乳杆菌高浓度组(1千克/吨饲料)均产生了H5抗体， 且时间依赖性的升高，但低浓度和高浓度组各时间点相比，差异不显著。
(2)H7抗体效价(log2)
表2-H7抗体效价(log2)
以上表2结果表明：和ATCC393干酪乳杆菌组(1千克/吨饲料)相比， H7N9-NP/ATCC393干酪乳杆菌低浓度(0.5千克/吨饲料)和H7N9-NP/ATCC393 干酪乳杆菌高浓度(1千克/吨饲料)均产生了H7抗体，且时间依赖性的升高， 但低浓度和高浓度组各时间点相比，差异不显著。
(3)H9抗体效价(log2)
表3-H9抗体效价(log2)
以上表3结果表明：和ATCC393干酪乳杆菌组(1千克/吨饲料)相比， H9N2-HA/ATCC393干酪乳杆菌低浓度组(0.5千克/吨饲料)和 H9N2-HA/ATCC393干酪乳杆菌高浓度组(1千克/吨饲料)均产生了H9抗体， 且时间依赖性的升高，但低浓度和高浓度组各时间点相比，差异不显著。
三、H5N6-HA/ATCC393干酪乳杆菌饲喂组血清样本中IgG亚类(IgG1、 IgG2a和IgG2b)抗体水平检测
用间接ELISA检测分离的血清样本中IgG亚类(IgG1、IgG2a和IgG2b)。
包被抗原均以1μg/mL浓度，每孔100μL包被96孔酶标板，37℃2h，然 后用PBST(PBS+吐温)洗涤。每孔加100μL 5％脱脂乳的PBST封闭液，37℃ 封闭1h，洗涤；每孔加入PBS稀释的待检免疫血清，37℃孵育1h，洗涤；每 孔加入PBS稀释的二抗，37℃孵育1h，洗涤；每孔加入100μLTMB显色液(3， 3'，5，5'-四甲基联苯胺)，室温显色10min后每孔加入50μL 2M的硫酸终止反 应，测OD450nm吸光值。分析结果。当样品OD450值≥(阴性血清的OD450 值+3倍标准方差)时即为阳性。
将各实验组抗原以1μg/孔包被好酶联免疫吸附板后，将一次饲喂后第4 周的血清按1:100比例稀释作为一抗，以HRP(辣根过氧化物酶)标记的羊抗 鸡IgG1、IgG2a、IgG2b以1:2000稀释后作为二抗，采用间接ELISA方法检测 抗体水平，酶标仪OD450下读取数值。
表4-鸡血清样本中IgG亚类(IgG1、IgG2a和IgG2b)OD450值
以上表4结果表明：H5N6-HA/ATCC393干酪乳杆菌低浓度(0.5千克/吨 饲料)和H5N6-HA/ATCC393干酪乳杆菌高浓度(1千克/吨饲料)产生的IgG1、 IgG2a和IgG2b抗体和ATCC393干酪乳杆菌组(1千克/吨饲料)相比，显著升 高，表明细胞免疫应答和体液免疫应答水平升高，低浓度和高浓度组相比，IgG1、IgG2a和IgG2b抗体水平差异不显著。
四、H7N9-HA/ATCC393干酪乳杆菌饲喂组血清样本中IgG亚类(IgG1、 IgG2a和IgG2b)抗体水平检测
用间接ELISA检测分离的血清样本中IgG亚类(IgG1、IgG2a和IgG2b)。
包被抗原均以1μg/mL浓度，每孔100μL包被96孔酶标板，37℃2h，然 后用PBST(PBS+吐温)洗涤。每孔加100μL 5％脱脂乳的PBST封闭液，37℃ 封闭1h，洗涤；每孔加入PBS稀释的待检免疫血清，37℃孵育1h，洗涤；每 孔加入PBS稀释的二抗，37℃孵育1h，洗涤；每孔加入100μLTMB显色液(3， 3'，5，5'-四甲基联苯胺)，室温显色10min后每孔加入50μL2M的硫酸终止反 应，测OD450nm吸光值。分析结果。当样品OD450值≥(阴性血清的OD450 值+3倍标准方差)时即为阳性。
将各实验组抗原以1μg/孔包被好酶联免疫吸附板后，将一次饲喂后第4 周的血清按1:100比例稀释作为一抗，以HRP(辣根过氧化物酶)标记的羊抗 鸡IgG1、IgG2a、IgG2b以1:2000稀释后作为二抗，采用间接ELISA方法检测 抗体水平，酶标仪OD450下读取数值。
表5-鸡血清样本中IgG亚类(IgG1、IgG2a和IgG2b)OD450值
以上表5结果表明：H7N9-HA/ATCC393干酪乳杆菌低浓度(0.5千克/吨 饲料)和H7N9-HA/ATCC393干酪乳杆菌高浓度(1千克/吨饲料)产生的IgG1、 IgG2a和IgG2b抗体和ATCC393干酪乳杆菌组(1千克/吨饲料)相比，显著升 高，表明细胞免疫应答和体液免疫应答水平升高，低浓度和高浓度组相比，IgG1、IgG2a和IgG2b抗体水平差异不显著。
五、H9N2-HA/ATCC393干酪乳杆菌饲喂组血清样本中IgG亚类(IgG1、 IgG2a和IgG2b)抗体水平检测
用间接ELISA检测分离的血清样本中IgG亚类(IgG1、IgG2a和IgG2b)。
包被抗原均以1μg/mL浓度，每孔100μL包被96孔酶标板，37℃2h，然 后用PBST(PBS+吐温)洗涤。每孔加100μL 5％脱脂乳的PBST封闭液，37℃ 封闭1h，洗涤；每孔加入PBS稀释的待检免疫血清，37℃孵育1h，洗涤；每 孔加入PBS稀释的二抗，37℃孵育1h，洗涤；每孔加入100μLTMB显色液(3， 3'，5，5'-四甲基联苯胺)，室温显色10min后每孔加入50μL2M的硫酸终止反 应，测OD450nm吸光值。分析结果。当样品OD450值≥(阴性血清的OD450 值+3倍标准方差)时即为阳性。
将各实验组抗原以1μg/孔包被好酶联免疫吸附板后，将一次饲喂后第4 周的血清按1:100比例稀释作为一抗，以HRP(辣根过氧化物酶)标记的羊抗 鸡IgG1、IgG2a、IgG2b以1:2000稀释后作为二抗，采用间接ELISA方法检测 抗体水平，酶标仪OD450下读取数值。
表6-鸡血清样本中IgG亚类(IgG1、IgG2a和IgG2b)OD450值
以上表6结果表明：H9N2-HA/ATCC393干酪乳杆菌低浓度(0.5千克/吨 饲料)和H9N2-HA/ATCC393干酪乳杆菌高浓度(1千克/吨饲料)产生的IgG1、 IgG2a和IgG2b抗体和ATCC393干酪乳杆菌组(1千克/吨饲料)相比，显著升 高，表明细胞免疫应答和体液免疫应答水平升高，低浓度和高浓度组相比，IgG1、IgG2a和IgG2b抗体水平差异不显著。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于 此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到 变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应 以所述权利要求的保护范围为准。
SEQUENCE LISTING
<110> 张大生
<120> 三种表达禽流感病毒蛋白的重组乳酸菌及构建方法和应用
<130> 2010
<160> 12
<170> PatentIn version 3.3
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Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala
325 330 335
Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Met
340 345 350
Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn
355 360 365
Glu Asn Met Glu Ala Met Glu Ser Asn Thr Leu Glu Leu Arg Ser Arg
370 375 380
Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg
385 390 395 400
Ala Ser Ala Gly Gln Val Ser Val Gln Pro Thr Phe Ser Val Gln Arg
405 410 415
Asn Leu Pro Phe Glu Arg Ala Ile Ile Met Ala Ala Phe Thr Gly Asn
420 425 430
Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met
435 440 445
Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe
450 455 460
Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp
465 470 475 480
Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr
485 490 495
Asp Asn
<210> 5
<211> 579
<212> DNA
<213> 未知
<400> 5
atgggactat tcggggccat agcagggttt atagagggag gttggtcagg actagttgct 60
ggttggtatg ggttccagca ttcaaatgac caaggggttg gtatggcagc agatagagac 120
tcaacccaaa aggcaattga taaaataaca tccaaagtga ataatatagt cgacaaaatg 180
aacaagcagt atgaaatcat tgatcatgaa ttcagtgagg tagaaactag acttaacatg 240
atcaataata agattgatga tcaaatccag gatatatggg catataatgc agaattgcta 300
gttctgcttg aaaaccagaa aacactcgat gagcatgacg caaatgtaaa caatctatat 360
aataaagtaa agagggcgtt gggttctaat gcggtggaag atgggaaagg atgtttcgag 420
ctctaccaca aatgtgatga ccaatgcatg gagacaattc ggaacgggac ctacaacaga 480
aggaagtatc aagaggagtc aaaattagaa agacagagaa tagagggggt caagctggaa 540
tctgaaggaa cttacaaaat cctcaccatt tattcgtga 579
<210> 6
<211> 498
<212> PRT
<213> 未知
<400> 6
Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly
1 5 10 15
Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met
20 25 30
Val Ser Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys
35 40 45
Leu Ser Asp Asn Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu
50 55 60
Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu
65 70 75 80
Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile
85 90 95
Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp
100 105 110
Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp
115 120 125
Ala Thr Ala Gly Leu Thr His Leu Met Ile Trp His Ser Asn Leu Asn
130 135 140
Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp
145 150 155 160
Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser
165 170 175
Gly Ala Ala Gly Ala Ala Val Lys Gly Ile Gly Thr Met Val Met Glu
180 185 190
Leu Ile Arg Met Ile Lys Arg Gly Val Asn Asp Arg Asn Phe Trp Arg
195 200 205
Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn
210 215 220
Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp
225 230 235 240
Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu
245 250 255
Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His
260 265 270
Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly
275 280 285
Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe
290 295 300
Arg Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu
305 310 315 320
Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala
325 330 335
Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Met
340 345 350
Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn
355 360 365
Glu Asn Met Glu Ala Met Glu Ser Asn Thr Leu Glu Leu Arg Ser Arg
370 375 380
Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg
385 390 395 400
Ala Ser Ala Gly Gln Val Ser Val Gln Pro Thr Phe Ser Val Gln Arg
405 410 415
Asn Leu Pro Phe Glu Arg Ala Ile Ile Met Ala Ala Phe Thr Gly Asn
420 425 430
Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met
435 440 445
Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe
450 455 460
Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp
465 470 475 480
Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr
485 490 495
Asp Asn
<210> 7
<211> 28
<212> DNA
<213> 人工序列
<400> 7
gcggtcgaca tggcgtctca aggcacca 28
<210> 8
<211> 27
<212> DNA
<213> 人工序列
<400> 8
cgcgatatct taattgtcat actcctc 27
<210> 9
<211> 28
<212> DNA
<213> 人工序列
<400> 9
gcggtcgaca tggcgtccca aggcacca 28
<210> 10
<211> 28
<212> DNA
<213> 人工序列
<400> 10
cgcgatatct taattgtcat actcctct 28
<210> 11
<211> 29
<212> DNA
<213> 人工序列
<400> 11
gcggtcgaca tgggactatt cggggccat 29
<210> 12
<211> 27
<212> DNA
<213> 人工序列
<400> 12
cgcgatatct cacgaataaa tggtgag 27
Claims (10)
1.表达禽流感病毒蛋白的重组乳酸菌菌株，其特征在于，含有编码H5N6禽流感病毒NP蛋白的基因序列、编码H7N9禽流感病毒NP蛋白的基因序列或编码H9N2禽流感病毒HA蛋白的基因序列中的任意一种。
2.根据权利要求1所述的表达禽流感病毒蛋白的重组乳酸菌菌株，其特征在于，所述编码H5N6禽流感病毒NP蛋白的基因序列如SEQ ID NO:1所示，所述H5N6禽流感病毒NP蛋白氨基酸序列如SEQ ID NO:2所示；所述编码H7N9禽流感病毒NP蛋白的基因序列如SEQ ID NO:3所示，所述H7N9禽流感病毒NP蛋白氨基酸序列如SEQ ID NO:4所示；所述编码H9N2禽流感病毒HA蛋白的基因序列如SEQ ID NO:5所示，所述H9N2禽流感病毒HA蛋白氨基酸序列如SEQID NO:6所示。
3.一种权利要求1-2任一项所述的表达禽流感病毒蛋白的重组乳酸菌菌株的构建方法，其特征在于，包括以下步骤：
(1)提取禽流感病毒RNA，进行反转录，得到cDNA；合成相应病毒基因的扩增引物，得到引物组；
(2)用所述cDNA和所述引物组进行PCR扩增，得到扩增产物，将所述扩增产物进行琼脂糖凝胶回收，得到目的基因；
(3)将所述目的基因与载体进行同源重组，得到同源重组产物；将所述同源重组产物进行克隆，挑取阳性克隆进行测序验证，得到重组质粒；
(4)将所述重组质粒导入ATCC393干酪乳杆菌感受态中，之后进行涂板、培养、挑取单克隆菌株进行鉴定，得到所述表达禽流感病毒蛋白的重组乳酸菌菌株。
4.根据权利要求3所述的表达禽流感病毒蛋白的重组乳酸菌菌株的构建方法，其特征在于，步骤(1)中，所述病毒为H5N6禽流感病毒、H7N9禽流感病毒或H9N2禽流感病毒中的任意一种。
5.根据权利要求4所述的表达禽流感病毒蛋白的重组乳酸菌菌株的构建方法，其特征在于，所述病毒为H5N6禽流感病毒时，合成NP基因的引物组H5N6-NP-1077F和H5N6-NP-1077R，序列如SEQ ID NO:7和SEQ ID NO:8所示；所述病毒为H7N9禽流感病毒时，合成NP基因的引物组H7N9-NP-1515F和H7N9-N9-1515R，序列如SEQ ID NO:9和SEQ ID NO:10所示；所述病毒为H9N2禽流感病毒时，合成HA基因的引物组H9N2-HA-579F和H9N2-HA-579R，序列如SEQ ID NO:11和SEQ ID NO:12所示；所述扩增引物上游含有SalI位点，下游含EcoRV位点。
6.根据权利要求3所述的表达禽流感病毒蛋白的重组乳酸菌菌株的构建方法，其特征在于，步骤(3)中，所述载体为pVE5523载体；
步骤(4)中，所述导入操作为：将所述重组质粒与ATCC393干酪乳杆菌感受态轻柔混匀后，放置冰上5min，之后转入直径1mm的预冷电转化杯中，迅速电击，电击参数为电压2.5kV，电击时间5ms；电击后加入800μL冰预冷的MRS培养基，混匀之后转移至1.5ml离心管中，放置冰上10min，28℃培养4h，得到菌液；
所述涂板和培养操作为：取100μL所述菌液涂布于含有5μg/ml红霉索的MRS固体培养基上，28℃培养3天；挑取单个菌落，分别接种于含有5ug/ml红霉素的MRS液体培养基中，28℃培养48h。
7.权利要求1-2任一项所述的表达禽流感病毒蛋白的重组乳酸菌菌株在制备预防和治疗禽流感药物中的应用，优选的，所述药物为口服疫苗。
8.一种预防和治疗禽流感的药物，其特征在于，包含权利要求1-2任一项所述的表达禽流感病毒蛋白的重组乳酸菌菌株。
9.一种制备权利要求8所述预防和治疗禽流感药物的方法，其特征在于，包括以下步骤：用MRS液体培养基将所述重组乳酸菌在37℃下培养72h，离心得到菌体，将所述菌体与辅料混匀后35℃干燥，得到粉剂；检测所述粉剂活菌数，保证活菌数>100亿/克，得到所述预防和治疗禽流感的药物。
10.根据权利要求9所述预防和治疗禽流感药物的制备方法，其特征在于，所述辅料包括50-80％的可溶性淀粉、5-20％脱脂乳、2-10％海藻糖、5-10％谷氨酸钠、1-5％山梨醇和1-5％维生素C。
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