CN110747219A - 一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗 - Google Patents
一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗 Download PDFInfo
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Abstract
本发明公开了一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,通过芽孢表面展示技术将鲤春病毒血症病毒的G蛋白展示在芽孢表面,通过富集培养和芽孢诱导获得重组菌的芽孢,再经冷冻干燥制备出固态的重组疫苗。本发明可以通过添加到饲料中进行直接投喂,来避免或降低鲤鱼鲤春病毒病的暴发,对于鲤鱼鲤春病毒血症具有较好的免疫预防作用。
Description
技术领域
本发明涉及鲤疾病免疫防治技术领域,特别涉及一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗。
背景技术
鲤鱼俗称鲤拐子、毛子等,隶属于鲤科,是我国主要的淡水鱼种之一,占我国淡水鱼类总量的一半以上。其身体侧扁而腹部圆,口呈马蹄形,须2对。背鳍基部较长,背鳍和臀鳍均有一根粗壮带锯齿的硬棘。体侧金黄色,尾鳍下叶橙红色。鲤鱼平时多栖息于江河、湖泊、水库、池沼的水草丛生的水体底层,以食底栖动物为主。因鲤鱼含有丰富的营养物质,具有降低胆固醇、防治动脉硬化、冠心病的功效,在中国和日本当做观赏鱼或食用鱼,在德国等欧洲国家作为食用鱼被养殖。鲤鱼经人工培育的品种很多,如红鲤、团鲤、草鲤、锦鲤、火鲤、芙蓉鲤、荷包鲤等,约有2900种。
鲤鱼养殖过程中病害频发,如烂鳃病、鳞立病、肠炎病等,这其中由鲤春病毒引起的鲤春病毒病(Spring viremia of carp简称SVC)尤为严重。鲤春病毒血症,即鲤鱼传染性腹水症,主要感染鲤鱼,同时对鲢鱼、鳙鱼、欧鲫、草鱼等具有感染性,可导致发病死亡,被OIE列为需申报疫病。由于此病毒只在春天气温上升时致病,故称鲤春病毒病,是一种急性、出血性、传染性病毒病,经常在鲤科鱼类特别是鲤、锦鲤中流行,引起幼鱼和成鱼死亡,危害严重。鲤春病毒病是国际兽医局(OIE)和我国进出境动物必检二类传染病,因此要做好养殖生产中的病害防治工作。然而,由于鲤鱼病毒生物学相关的基础研究工作相对滞后,加上养殖环境因子的复杂性,无法对引起鲤鱼鲤春病毒血症的致病机制进行判定,导致该病毒病时有爆发,给养殖户带来极大地经济损失。
发明内容
本发明的目的在于提供一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,可以通过添加到饲料中进行直接投喂,来避免或降低鲤鱼鲤春病毒病的暴发,对于鲤鱼鲤春病毒血症具有较好的免疫预防作用。
本发明解决其技术问题所采用的技术方案是:
一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,通过以下步骤制备而得:
(1)病毒抗原的筛选
选择鲤春病毒血症病毒的G蛋白作为构建核酸疫苗的抗原;
(2)G蛋白基因的克隆表达
通过PCR的方法从鲤春病毒血症病毒中扩增获得G蛋白基因;扩增的G蛋白基因克隆于pMD18-T载体中,然后转化大肠杆菌DH5α筛选出阳性克隆菌株;经Nco I、Sac I双酶切鉴定后,连接到中间桥梁质粒pET30a-G,再利用Sac I、Kpn I进行双酶切后,将目的基因克隆到质粒pJS700载体上,构建整合型重组质粒pJS700-G;
(3)G蛋白的枯草芽孢表面展示
将整合型重组质粒pJS700-G转化感受态细胞,得到重组菌株;
(4)重组芽孢杆菌的培养
将重组菌株在DSM培养基中进行培养,诱导产生芽孢,对芽孢进行冷冻干燥后,保存于-80℃待用。
鲤春病毒血症是由一种弹状病毒即鲤春病毒血症病毒(SVCV)引起鲤鱼科的一种急性出血性传染病,SVCV基因组为线性的,单股不分段的负链RNA,主要包含五个开放性阅读框,分别编码五种蛋白质:核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和RNA聚合酶(L)。发明人经过试验探索,选择SVCV G蛋白作为构建核酸疫苗的抗原能取得较好效果。
本发明针对目前鲤鱼产业中危害严重的鲤春病毒血症,受多种因素影响,目前该病发生率居高不下,发病诱因复杂,且市场暂时无显著疗效鱼药可用,所以疫苗接种免疫是一个较好的途径。市面上常见的商品化疫苗主要为灭活苗和弱毒苗。灭活疫苗又称死疫苗,是指利用加热或甲醛等理化方法将人工大量培养的完整的病原微物杀死,使其丧失感染性和毒性而保持免疫原性,并结合相应的佐剂而制成的疫苗。由于灭活疫苗需要用到大量发病鱼组织或细胞,存在着成本高、有潜在感染危险等缺陷,很难在实践中推广应用。发明人根据筛选到的这种芽孢杆菌表面展示载体,本发明研制了一种成本低、适宜推广应用的口服疫苗,满足养殖过程中鲤鱼鲤春病毒血症的防疫需要,以期降低鲤鱼养殖过程中的发病率和死亡率。
鲤鱼鲤春病毒血症口服疫苗的使用方法为:在饲料加工过程中,直接将鲤鱼鲤春病毒血症口服疫苗加入到饲料原料中混匀,制备成带疫苗的饲料投喂。
鲤鱼鲤春病毒血症口服疫苗的使用方法为:在饲料投喂前,将鲤鱼鲤春病毒血症口服疫苗化于水中,喷洒在饲料上,待饲料吸附干燥后投喂。
所述感受态细胞为Bacillus subtilis感受态细胞。
通过PCR的方法从鲤春病毒血症病毒中扩增获得G蛋白基因采用的引物如下:
正向引物:5’-CATGCCATGGATGTCTATCATCAGCTACATCGCA-3’,
反向引物:5’-CGAGCTCAACGAAGGACCGCATTTCG-3’。
本发明的有益效果是:本发明根据枯草芽孢杆菌芽孢耐高温、可调节水质的特性,通过芽孢表面展示技术将鲤春病毒血症病毒的G蛋白展示在芽孢表面。通过富集培养和芽孢诱导获得重组菌的芽孢,再经冷冻干燥制备出固态的重组疫苗,根据免疫保护需要,添加不同浓度的重组疫苗到饲料中,克服现有口服疫苗无法耐高温、无法添加到饲料中去的弊端,有利于鲤鱼对目的抗原的吸收,适合鲤鱼鲤春病毒血症的筛查与预防。
附图说明
图1为整合型质粒构建图。
图2为鲤鱼鲤春病毒G蛋白重组蛋白表达图。M:蛋白质Marker,48KDa、63KDa为所指处条带蛋白大小,58.59KDa为目的蛋白;1:E.coli.BL21(DE3)pET-30a空质粒;2:未诱导;3-7:加IPTG诱导终浓度分别为1.0、0.8、0.6、0.4、0.2mM。
图3为重组芽孢杆菌芽孢冻干粉的菌落计数图。上面一排平板是冻干粉稀释1011倍的3个重复,下面一排平板是冻干粉稀释1010倍的3个重复,芽孢浓度取平均数。
图4为不同种浓度重组芽孢杆菌预混入成品饲料后取样培养的菌浓度分析图。空白组:未加任何芽孢杆菌的饲料;野生型:添加109cpu/ml野生型芽孢杆菌的饲料;试验1:添加109cpu/ml重组芽孢杆菌的饲料;试验2:添加1010cpu/ml重组芽孢杆菌的饲料;试验3:添加1011cpu/ml重组芽孢杆菌的饲料。
图5口服疫苗免疫后不同时段的血清抗体水平TAS-ELISA试验。1、PSB对照;2、口服免疫组1:连续免疫14天;3、口服免疫组2:连续投喂7天后停3天,重复3次。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。
本发明中,若非特指,所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
材料
鲤春病毒血症病毒(SVCV)由浙江省淡水水产研究所鱼病研究室提供;正常鲤鱼购自浙江省金华市某养殖场;所用芽孢杆菌、表达载体由江苏大学生科院提供;各种酶、PCR试剂、培养基购自Takara公司;引物合成、其他耗材购自上海生工生物公司。
实施例:
一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,通过以下步骤制备而得:
(1)鲤春病毒血症病毒G蛋白基因的克隆表达:通过PCR的方法从保存的SVCV病毒中扩增获得G蛋白基因;反应体系:2×PCR Master Mix 25μl,10μM引物(SVG-F:5’-CATGCCATGGATGTCTATCATCAGCTACATCGCA-3’(SEQ ID No.1),SVG-R:5’-CGAGCTCAACGAAGGACCGCATTTCG-3’(SEQ ID No.2)各1μl,模板3μl,用双蒸水补足至50μl;扩增反应条件:95℃预变性4min;94℃变性45s、55℃退火45s、72℃延伸1min,35个循环后72℃10min)。扩增片段克隆于pMD18-T载体中,然后转化大肠杆菌DH5α筛选出阳性克隆菌株。经Nco I、Sac I双酶切鉴定后,连接到中间桥梁质粒pET30a-G,再利用内切酶Sac I、Kpn I进行双酶切后,将目的基因克隆到质粒pJS700载体上,构建整合型重组质粒pJS700-G(图1和2)。
(2)G蛋白的枯草芽孢表面展示:挑Bacillus subtilis单菌落至3ml LB培养基中,37℃,250r/min培养过夜;第二天取10%培养液转接至3ml SPI培养基中,37℃,250r/min培养4-5h;取5%的培养液至3ml SPII培养基中,37℃,100r/min培养90分钟,即得到感受态细胞。将1μL整合型重组质粒pJS700-G转化到Bacillus subtilis感受态细胞,通过含1%淀粉的LB平板筛选,得到重组菌株Bacillus subtilis/pJS700-G。
(3)重组芽孢杆菌的培养:将菌株在DSM培养基中进行培养,37℃48小时,诱导产生芽孢,10000rpm15min离心收集沉淀,对芽孢进行冷冻干燥后,进行浓度分析鉴定(图3),保存于-80℃待用。
疫苗的使用
(1)含疫苗饲料的制备:A:在饲料加工过程中,直接将浓度分别为109cpu/ml、1010cpu/ml、1011cpu/ml重组芽孢杆菌10ml经稀释后加入到10Kg原料中,混匀后在饲料加工机中经80-100℃高温加热压缩制备成带不同浓度疫苗的成品饲料3种,对成品饲料中的芽孢杆菌存活率进行分析,芽孢杆菌存活率与重组芽孢杆菌浓度呈正相关(图4),因此,选择含有高浓度重组芽孢杆菌的饲料备用;B:购置成品饲料,在饲料投喂前,将10ml 1011cpu/ml浓度的疫苗化于水中,喷洒在10Kg饲料上,待饲料吸附干燥后投喂。
(2)按照不同大小的鲤鱼每日平均摄食量,分别采取2种投喂方案:A:连续投喂2周(标记为“口服免疫组1”);B:连续投喂带疫苗饲料7天,再投喂无疫苗饲料3天,重复3次后正常投喂(标记为“口服免疫组2”);C:PBS为对照组;
鲤鱼口服免疫效果评估
(1)随机挑选免疫后28天的鲤鱼和PBS对照组各50只,利用病毒SVCV进行人工感染,进行相对免疫保护率(Relative percent survival,RPS)比较,其中RPS=(对照组死亡率—免疫组死亡率)/对照组死亡率×100%;
(2)攻毒后继续饲养观察10天,统计鲤鱼存活数和相对保护率。
利用本发明发现的一种鲤鱼鲤春病毒血症病原——鲤春病毒(SVCV),经过病原回感试验,采用口服灭活疫苗免疫的方式进行阶段性免疫后,分不同的时间段采集鲤鱼血清,利用TAS-ELISA试验分析了免疫后鲤鱼血清相应抗体的变化,结果表明鲤鱼血清中目的抗体显著提高(图5);相对免疫保护率试验表明,口服疫苗后鲤鱼在病原菌感染后存活率明显提高(表1)。
表1口服疫苗相对免疫保护率试验结果
组别 | 攻毒数(只) | 存活数(只) | 相对保护率(%) |
PBS | 50 | 9 | 0 |
口服免疫组1 | 50 | 33 | 66% |
口服免疫组2 | 50 | 39 | 78% |
根据以上实验结果表明,本发明所制备的口服疫苗能够有效地提高鲤鱼养殖过程中抗鲤春病毒的感染能力,能够显著降低鲤春病毒血症的爆发。
以上所述的实施例只是本发明的一种较佳的方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
SEQUENCE LISTING
<110> 浙江省淡水水产研究所
<120> 一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗
<130> 2019.11.29
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 34
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
catgccatgg atgtctatca tcagctacat cgca 34
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
cgagctcaac gaaggaccgc atttcg 26
Claims (5)
1.一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,其特征在于,通过以下步骤制备而得:(1)病毒抗原的筛选
选择鲤春病毒血症病毒的G蛋白作为构建核酸疫苗的抗原;
(2)G蛋白基因的克隆表达
通过PCR的方法从鲤春病毒血症病毒中扩增获得G蛋白基因;扩增的G蛋白基因克隆于pMD18-T载体中,然后转化大肠杆菌DH5α筛选出阳性克隆菌株;经Nco I、Sac I双酶切鉴定后,连接到中间桥梁质粒pET30a-G,再利用Sac I、Kpn I进行双酶切后,将目的基因克隆到质粒pJS700载体上,构建整合型重组质粒pJS700-G;
(3)G蛋白的枯草芽孢表面展示
将整合型重组质粒pJS700-G转化感受态细胞,得到重组菌株;
(4)重组芽孢杆菌的培养
将重组菌株在DSM培养基中进行培养,诱导产生芽孢,对芽孢进行冷冻干燥后,保存于-80℃待用。
2.根据权利要求1所述的一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,其特征在于,使用方法为:在饲料加工过程中,直接将鲤鱼鲤春病毒血症口服疫苗加入到饲料原料中混匀,制备成带疫苗的饲料投喂。
3.根据权利要求1所述的一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,其特征在于,使用方法为:在饲料投喂前,将鲤鱼鲤春病毒血症口服疫苗化于水中,喷洒在饲料上,待饲料吸附干燥后投喂。
4.根据权利要求1所述的一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,其特征在于,所述感受态细胞为Bacillus subtilis感受态细胞。
5.根据权利要求1所述的一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,其特征在于,通过PCR的方法从鲤春病毒血症病毒中扩增获得G蛋白基因采用的引物如下:
正向引物:5’-CATGCCATGGATGTCTATCATCAGCTACATCGCA-3’,
反向引物:5’-CGAGCTCAACGAAGGACCGCATTTCG-3’。
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