CN111000995A - 猪用三联灭活疫苗及其制备方法和应用 - Google Patents
猪用三联灭活疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了猪用三联灭活疫苗及其制备方法和应用。所述猪用三联灭活疫苗包括:表达O型FMDV VP1基因的重组杆状病毒、猪塞内卡病毒、H3N2亚型猪流感病毒和免疫佐剂。本发明还公开了制备所述猪用三联灭活疫苗的方法,包括:(1)将表达O型FMDV VP1基因的重组杆状病毒扩增后灭活;(2)将灭活的表达O型FMDV VP1基因的重组杆状病毒液和灭活的猪塞内卡病毒液以及H3N2亚型猪流感病毒混合得到水相;(3)将免疫佐剂加热得到油相;(4)将油相加入到水相中混合,乳化。免疫保护效力检测结果证明本发明所制备的猪用三联灭活疫苗能够同时有效防治O型***病毒、猪塞内卡病毒和H3N2亚型猪流感病毒。
Description
技术领域
本发明涉及猪用三联灭活疫苗,尤其涉及猪***杆状病毒载体、猪塞内卡病毒和H3N2亚型猪流感病毒三联灭活疫苗及其制备方法,属于猪用三联灭活疫苗领域。
背景技术
O型***病毒是***病毒属、小RNA病毒科的成员之一。在所有七种***病毒血清型中,O型在全世界范围内流行和分布最广,非洲北部、东欧、整个南美洲以及亚洲的大部分地区都有报道,严重威胁全球畜牧业旳发展。灭活苗在制备过程中因设备安全性要求高、工艺复杂、花费大、毒株灭活不完全、有散毒的危险等缺点,因此杆状病毒载体疫苗被认为是***病毒疫苗研究领域最具潜力的候选疫苗,利用基因重组技术合成并表达具有病毒抗原表位的病毒样颗粒能够诱导动物产生近乎病毒自然感染的免疫力,一直受到高度重视。
塞内卡谷病毒最早由研究者从人胚胎视网膜细胞PER.C6的细胞培养污染物中分离得到,且其被认为源自培养细胞使用的牛血清或者猪源胰蛋白酶。2015年,巴西学者从患有水泡性疾病猪只的水泡液和血清中检测到SVA全基因组的存在,并认为SVV感染与猪的特发性水泡病相关。随后美国、巴西、加拿大、中国、泰国等全球多个国家陆续报道了猪感染SVV的案例。SVV可造成不同年龄阶段的猪群感染,主要临床表现为蹄部和口鼻部发生水泡、溃疡,感染新生仔猪可引起死亡。塞内卡病毒目前尚无商品化的疫苗生产。
流感病毒属于正黏病毒科,1918年首次发现于美国,引起一种急性、发热性、高度接触感染性的呼吸道传染病。猪流行性感冒的高发季节多在深秋、寒冬直至早春,短时内爆发性流行。猪感染S IV后的典型临床症状表现为咳嗽、喷嚏、流涕、高热、食欲减退、精神沉郁、衰竭、呼吸困难、延迟出栏、繁殖障碍等,其特点表现为传染性强、发病率高,死亡率较低,但会严重影响猪的生长性能,延迟出栏时间;同时猪流行性感冒极易与其它呼吸道疾病混合或继发感染,发展成猪呼吸道疾病综合征,使死亡率骤增,给养猪业造成极大危害。
迄今为止,缺乏一种能够同时有效防治O型***病毒、猪塞内卡谷病毒和流感病毒的猪用三联灭活疫苗。
发明内容
本发明的目的之一是提供同时有效防治O型***病毒、猪塞内卡谷病毒和流感病毒的猪用三联灭活疫苗
本发明的目的之二是提供一种制备所述猪用三联灭活疫苗的方法;
本发明的上述目的是通过以下技术方案来实现的:
一种猪用三联灭活疫苗,包括:表达O型FMDV VP1基因的重组杆状病毒、猪塞内卡病毒、H3N2亚型猪流感病毒和免疫佐剂。
所述表达O型FMDV VP1基因的重组杆状病毒的构建方法包括:。
(1)将O型FMDV VP1基因克隆至pFastBacⅠ载体得到重组质粒pFB-FMDV-vp1;(2)用重组质粒pFB-FMDV-vp1构建得到重组穿梭杆状质粒rBacmid-FMDV-vp1;(3)将重组穿梭杆状质粒转染昆虫细胞得到表达O型FMDV VP1基因的重组杆状病毒。
为了提高将O型FMDV VP1基因的表达效率,可以将O型FMDV VP1基因进行密码子优化以提高其在昆虫宿主细胞中的表达效率;其中,O型FMDV VP1基因的原始序列是SEQ IDNO.1所示,密码子优化后的序列为SEQ ID NO.2所示;密码子优化后的序列在昆虫细胞中的表达效率相比原始序列有非常显著的提升。
其中,所述的昆虫细胞优选为Sf9细胞。
本发明中所述的猪塞内卡病毒优选为微生物保藏编号为:CGMCC No.18851的猪塞内卡病毒。
本发明中所述的H3N2亚型流感病毒优选为微生物保藏编号为:CGMCC No.14740的流感病毒。
本发明进一步提供了一种制备所述猪用三联灭活疫苗的方法,包括:
(1)将表达O型FMDV VP1基因的重组杆状病毒进行扩增后灭活;将猪塞内卡病毒进行扩增后灭活;(2)将灭活的表达O型FMDV VP1基因的重组杆状病毒液和灭活的猪塞内卡病毒液以及灭活的H3N2亚型流感病毒液混合均匀得到水相;(3)将免疫佐剂加热得到油相;(4)将油相加入到水相中混合,乳化,即得。
为了取得更好的效果,步骤(2)中将灭活的FMDV-vp1重组杆状病毒液、猪塞内卡病毒液和H3N2亚型流感病毒液混合后使使每头份疫苗中FMDV-vp1蛋白含量不小于40μg、猪塞内卡病毒不小于108.0TCID50、H3N2亚型猪流感病毒不小于107.5EID50。
本发明中所述的免疫佐剂优选为法国赛比克公司MontanideTM206佐剂;其中,步骤(3)中优选将免疫佐剂加热至30℃得到油相。
步骤(4)中控制油相和水相的体积比为1:1;其中,先将水相加入乳化罐内慢速搅拌然后缓慢加入油相佐剂,加完后以800r/min搅拌30分钟,再静止30分钟。
灭活疫苗的免疫保护效力检验表明,采用本发明制备的猪用三联灭活疫苗免疫猪,猪免疫后每周采血,利用***O型抗体液相阻断ELISA检测试剂盒测定猪血清中FMDV抗体水平、采用中和试验方法测定血清中SVA中和抗体水平,采用中和试血凝抑制试验方法测定血清中和猪流感病毒HI抗体水平。根据检测结果可见,所有免疫猪的血清中均同时产生高水平的FMDV抗体、SVA抗体和SIA抗体,免疫保护效力检测结果证明本发明制备的猪用三联灭活疫苗能够同时防治O型***病毒、猪塞内卡病毒和H3N2亚型流感病毒。
附图说明
图1重组杆状病毒穿梭载体DNA的PCR鉴定;1:DNA分子标记DL2000;2:构建好重组杆状病毒穿梭载体;3:阴性对照。
具体实施方式
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1猪用三联灭活疫苗的制备
1毒种
猪塞内卡病毒,其微生物保藏编号为:CGMCC No.18851;分类命名:塞内卡病毒;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;保藏时间是2019年10月29日;保藏地址:北京市朝阳区北辰西路1号院3号。
H3N2亚型猪流感病毒,其微生物保藏编号为:CGMCC No.14740;分类命名是:H3N2亚型猪流感病毒;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;保藏时间是2017年11月16日;保藏地址:北京市朝阳区北辰西路1号院3号。
2试验方法
2.1猪***杆状病毒制备
2.1.1 FMDV-vp1基因的设计与合成
将O型FMDV VP1基因(SEQ ID NO.1)经密码子优化后得到优化后的序列(SEQ IDNO.2),在优化后的序列两端添加EcoRⅠ和Hind III酶切位点克隆至pFastBacⅠ载体,形成重pFB-FMDV-vp1。
2.1.2重组穿梭杆状质粒rBacmid-FMDV-vp1的构建与制备
2.1.2.1重组穿梭杆状质粒的构建
将1μL重组质粒pFB-FMDV-vp1加入至200μL于冰上融化的DH10Bac感受态细胞,轻轻混匀后冰浴30min,42℃热激45s,迅速置于冰上2min,加入900μL的S.O.C培养基,37℃220rpm振荡培养4h,然后用S.O.C培养基做10-1,10-2,10-3三个稀释度,每个稀释度取150μL均匀的涂布于LB选择性琼脂平板上,37℃温箱避光培养36-48h,直至清晰蓝白菌落出现。
2.1.2.2重组杆状病毒穿梭载体DNA的提取
将LB选择性平板放于4℃冰箱2h,使菌落充分显色,将平板取出进行蓝白斑的筛选,挑取平板上较大的散在白色单菌落,重新划线接种于LB选择性平板进行第二次的蓝白斑筛选,37℃过夜培养后,同样,挑取平板上较大的散在白色单菌落接种至含50μg/ml的卡那霉素,7μg/ml的庆大霉素,10μg/ml的四环霉素的LB液体培养基中37℃220rpm振荡培养16个小时,提取杆粒DNA。
2.1.2.3重组杆状病毒穿梭载体DNA的PCR鉴定
以提取的重组杆状病毒穿梭载体DNA为模板,利用表1的FMDV-vp1-R/FMDV-vp1-F引物进行PCR扩增。
表1 FMDV-vp1-R/FMDV-vp1-F引物序列
循环参数:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸10min,共设置30个循环;72℃延伸10min。预计PCR扩增片段长度约为630bp,验证正确的重组杆状质粒DNA命名为rBacmid-FMDV-vp1。
对构建的重组杆状病毒穿梭载体进行PCR鉴定,扩增片段长度为630bp(图1)。鉴定结果表明FMDV-vp1蛋白成功转化至DH10Bac感受态细胞。
根据测得的不同浓度标准品OD值,以OD值为纵坐标,以浓度为横坐标,绘制标准曲线,根据线性回归方程,计算FMDV-vp1蛋白浓度为1.6mg/ml。
2.1.3重组杆状质粒转染Sf9细胞
转染前选择状态良好的Sf9细胞铺于6孔板,取适量传代Sf9细胞使用台盼蓝染色30s左右,滴于血球计数板观察细胞活性并计数。将细胞活性≥97%的细胞铺于6孔板,每孔106个细胞左右。2h(或过夜)后细胞贴壁,可用于转染。转染过程如下(细胞传代、转染均在无菌条件下操作):
1、准备两个灭菌的1.5ml EP管,各分装0.1ml无抗生素、无胎牛血清的Sf-900培养基;
2、分别加入3μl重组杆粒(2μg)、6μl转染试剂,轻轻混匀,室温静置5min。
3、将含有转染试剂的培养基转移到含有杆粒的培养基中,轻轻混匀,室温静置20min;期间取出贴壁的Sf9细胞,弃去原培养基,使用无抗生素、无胎牛血清的Sf-900培养基轻轻洗两遍,每孔加入2ml无抗生素、无胎牛血清的Sf-900培养基;
4、将上述的209μl混合液均匀滴加到6孔板的一个细胞孔中,另一孔为正常细胞对照。转染第3~5天,观察细胞变化。
2.1.4重组杆状病毒的扩增及滴度测定
将状态良好的Sf9细胞悬液传至细胞瓶,细胞贴壁后,接种10μl P0代病毒液。接毒约72h,Sf9细胞开始出现病变,第5或6天,待细胞出现变大变圆、胞内颗粒状物非常明显时,收获病毒液,-80℃避光保存。
2.1.5 FMDV-vp1蛋白的测定
利用Bradford蛋白定量测定试剂盒,采用微孔酶标仪法,测定FMDV-vp1蛋白浓度,试验严格按照说明书进行。
2.1.6灭活及灭活检验
2.1.6.1灭活
收获的猪***杆状病毒液中缓慢加入β-丙内脂溶液,使其终浓度为0.05%,充分混匀,4℃灭活8小时,期间每两个小时搅拌一次,灭活后将病毒液置于37℃水解2小时。灭活后的病毒液置2~8℃保存。
2.1.6.2灭活检验
取灭活后的病毒液,接种于已形成良好单层的Sf9细胞,置27℃培养观察4日,反复冻融2次后,再盲传2代,观察细胞病变情况。
灭活后的猪***杆状病毒接种SF9细胞,并盲传2代,细胞均无病变产生,结果表明病毒液灭活完全。
2.2猪塞内卡病毒制备
2.2.1病毒增殖
将状态良好的BHK-21细胞悬液传至细胞瓶,按培养基总量的5‰将毒种接种长满单层的BHK-21细胞,置37℃5%CO2培养箱中培养,吸附1小时后,补加血清含量为2%的DMEM细胞培养液,继续在37℃5%CO2培养箱中培养。接毒后细胞培养40-48小时,当细胞病变达到80%以上时,收获细胞及细胞培养物。反复冻融2次后,使用TCID50方法检测病毒滴度。
猪塞内卡病毒病毒含量测定:将猪塞内卡病毒种毒接种长满单侧的BHK-21细胞,培养44小时收获,反复冻融2次,测定病毒含量为108.7TCID50/ml。
2.2.2病毒灭活及检验
2.2.1.1病毒灭活
向收获的猪塞内卡病毒缓慢加入β-丙内脂溶液,使其终浓度为0.05%,充分混匀,4℃灭活8小时,期间每两个小时搅拌一次,灭活后将病毒液置于37℃水解2小时。灭活后的病毒液置2~8℃保存。
2.2.1.2病毒灭活检验
取灭活后的抗原液,按培养液总量的1‰接种到长满单层的BHK-21细胞,置37℃下培养48小时。冻融2次后,接种细胞盲传2代,观察细胞病变情况。灭活后的猪塞内卡病毒接种BHK-21细胞,并盲传2代,细胞均无病变产生,结果表明病毒液灭活完全。
2.3H3亚型猪流感病毒制备
2.3.1病毒增殖
将状态良好的MDCK细胞悬液传至细胞瓶,按培养基总量的1%将毒种接种长满单层的MDCK细胞,添加TPCK-胰酶,使其终浓度为2.5μg/ml,补加血清含量为2%的DMEM细胞培养液,置37℃5%CO2培养箱中培养,接毒后细胞培养72小时,当细胞病变达到80%以上时,收获细胞及细胞培养物。反复冻融2次后,使用EID50方法检测病毒滴度。
2.3.2病毒灭活
向收获的猪流感病毒缓慢加入β-丙内脂溶液,使其终浓度为0.08%,充分混匀,4℃灭活10小时,期间每两个小时搅拌一次,灭活后将病毒液置于37℃水解2小时。灭活后的病毒液置2~8℃保存。
2.3.4病毒灭活检验
取灭活后的猪流感病毒,按培养液总量的1%接种到长满单层的MDCK细胞,置37℃下培养72小时。冻融2次后,接种细胞盲传2代,观察细胞病变情况。
2.4灭活疫苗的配制
2.4.1油相制备取法国赛比克公司MontanideTM206佐剂,加热至30℃。
2.4.2水相制备将FMDV-vp1重组杆状病毒液、猪塞内卡病毒液和猪流感病毒以一定的比例混合,使每头份疫苗中FMDV-vp1蛋白含量不小于40μg、猪塞内卡病毒不小于108.0TCID50、猪流感病毒不小于107.5EID50
2.4.3乳化水相与油相体积比为1:1。先将水相加入乳化罐内慢速搅拌,然后缓慢加入油相佐剂,加完后以800r/min搅拌30分钟,再静止30分钟。
试验例1猪用三联灭活疫苗的成品检验
1无菌检验
按现行《中国兽药典》附录迪对猪***杆状病毒、猪塞内卡病毒和H3亚型猪流感病毒三联灭活疫苗检验。结果表明成品无菌检验合格。
2安全检验
取8周龄健康易感猪5头,每只颈部肌肉接种猪***杆状病毒载体、猪塞内卡病毒、H3亚型猪流感病毒三联灭活疫苗6ml,观察7日,观察接种疫苗后有无不良反应。疫苗接种5只健康易感猪,观察期间5只试验猪全部健活,无局部和全身不良反应。
试验例2猪用三联灭活疫苗的效力检验试验
1试验方法
筛选FMDV阴性、SVA阴性、SIV阴性8周龄健康易感猪10头,试验猪随机分为2组,每组5只。第1组,每只颈部肌肉接种疫苗3ml,接种3周后同样方式进行加强免疫1次。第2组不免疫,作空白对照。
1.1 FMDV部分
首免后每周采血分离血清,用***O型抗体液相阻断ELISA检测试剂盒测定猪血清中FMDV抗体水平。
1.2 SVA部分
首免后每周采血分离血清,用中和试验方法测定猪血清中塞内卡病毒中和抗体水平。
1.3 SIA部分
首免后每周采血分离血清,用中和试验方法测定猪血清中H3亚型猪流感HI抗体效价。
2检验结果
2.1 FMDV特异性抗体水平
猪免疫后每周采血,利用中国农业科学院兰州兽医研究所生产的***O型抗体液相阻断ELISA检测试剂盒测定猪血清中FMDV抗体水平(表1)。结果表明,与对照组相比,所有免疫组的抗体水平在第4或第5周时达到最高,最高达到1:512-1:1024之间,且达到最高后第6周抗体水平几乎不变。
表1血清中FMDV特异性抗体水平
注:“-”代表
2.2SVA中和抗体水平
猪免疫后每周采血,采用中和试验方法测定血清中SVA中和抗体水平(表2)。结果表明,与对照组相比,所有免疫组的抗体水平在第4或第5周时达到最高,最高达到1:512-1:2048之间,且达到最高后第6周抗体水平几乎不变。
表2血清中SVA中和抗体水平
2.3H3亚型猪流感病毒HI抗体水平
猪免疫后每周采血,采用中和试血凝抑制试验方法测定血清中和猪流感病毒HI抗体水平(表3)。结果表明,与对照组相比,所有免疫组的抗体水平在第5或第6周时达到最高,最高达到1:640-1:2560之间。
表3血清中H3亚型猪流感病毒HI抗体水平
SEQUENCE LISTING
<110> 哈药集团生物疫苗有限公司
<120> 猪用三联灭活疫苗及其制备方法和应用
<130> HLJ-3002-191203A
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Claims (10)
1.一种猪用三联灭活疫苗,其特征在于,包括:表达O型FMDV VP1基因的重组杆状病毒、猪塞内卡病毒、H3N2亚型猪流感病毒和免疫佐剂。
2.按照权利要求1所述的猪用三联灭活疫苗,其特征在于,所述表达O型FMDV VP1基因的重组杆状病毒的构建方法包括:
(1)将O型FMDV VP1基因克隆至pFastBacⅠ载体得到重组质粒pFB-FMDV-vp1;(2)用重组质粒pFB-FMDV-vp1构建得到重组穿梭杆状质粒rBacmid-FMDV-vp1;(3)将重组穿梭杆状质粒转染昆虫细胞,得到表达O型FMDV VP1基因的重组杆状病毒。
3.按照权利要求2所述的猪用三联灭活疫苗,其特征在于,所述的O型FMDV VP1基因是密码子优化后的基因,其核苷酸序列为SEQ ID NO.2所示;所述的昆虫细胞为Sf9细胞。
4.按照权利要求1所述的猪用三联灭活疫苗,其特征在于,所述猪塞内卡病毒的微生物保藏编号为:CGMCC No.18851。
5.按照权利要求1所述的猪用三联灭活疫苗,其特征在于,所述的H3N2亚型猪流感病毒微生物保藏编号为:CGMCC No.14740。
6.一种制备权利要求1所述猪用二联灭活疫苗的方法,包括:
(1)将表达O型FMDV VP1基因的重组杆状病毒进行扩增后灭活;将猪塞内卡病毒进行扩增后灭活;(2)将灭活的表达O型FMDV VP1基因的重组杆状病毒液、灭活的猪塞内卡病毒液以及灭活的H3N2亚型猪流感病毒液混合均匀得到水相;(3)将免疫佐剂加热得到油相;(4)将油相加入到水相中混合,乳化,即得。
7.按照权利要求6所述的方法,其特征在于,步骤(2)中将灭活的FMDV-vp1重组杆状病毒液和猪塞内卡病毒液混合后使使每头份疫苗中FMDV-vp1蛋白含量不小于40μg、猪塞内卡病毒不小于108.0TCID50、猪流感病毒不小于107.5EID50。
8.按照权利要求6所述的方法,其特征在于,所述的免疫佐剂为MontanideTM206佐剂。
9.按照权利要求6所述的方法,其特征在于,步骤(3)中将免疫佐剂加热至30℃得到油相。
10.按照权利要求5所述的方法,其特征在于,步骤(4)中控制油相和水相的体积比为1:1;所述的乳化包括:先将水相加入乳化罐内慢速搅拌然后缓慢加入油相佐剂,加完后以800r/min搅拌30分钟,再静止30分钟。
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