CN112641936B - 一种鹅星状病毒纤突蛋白脂质体疫苗及其制备方法和应用 - Google Patents
一种鹅星状病毒纤突蛋白脂质体疫苗及其制备方法和应用 Download PDFInfo
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- CN112641936B CN112641936B CN202110008346.6A CN202110008346A CN112641936B CN 112641936 B CN112641936 B CN 112641936B CN 202110008346 A CN202110008346 A CN 202110008346A CN 112641936 B CN112641936 B CN 112641936B
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Abstract
本发明公开了一种鹅星状病毒纤突蛋白脂质体疫苗,包括:脂质体,以及结合或包裹于脂质体中的VP27蛋白;脂质体由卵磷脂、胆固醇、抗氧化剂按薄膜扩散法制得。本发明还公开了上述鹅星状病毒纤突蛋白脂质体疫苗制备方法,包括如下步骤:将卵磷脂、胆固醇、抗氧化剂混合,溶于有机溶剂中,减压旋蒸除去有机溶剂,得到膜状空白脂质体;向膜状空白脂质体中加入磷酸盐缓冲液和玻璃珠,水平摇床进行水化得到混悬液;将混悬液分离,加入VP27蛋白溶液,超声处理得到鹅星状病毒纤突蛋白脂质体疫苗。本发明还公开了上述鹅星状病毒纤突蛋白脂质体疫苗在制备预防或治疗雏鹅痛风病的药物中的应用。
Description
技术领域
本发明涉及动物疫苗技术领域,尤其涉及一种鹅星状病毒纤突蛋白脂质体疫苗及其制备方法和应用。
背景技术
疫苗是预防和控制传染病最有效、最经济的手段,可追溯至最早应用于接种的牛痘,发展至今已有多种多样的疫苗。目前疫苗在禽类养殖生产中的应用已然是不可或缺的。传统疫苗包括灭活疫苗和弱毒疫苗,主要是通过灭活或减毒病原体来制备疫苗,但灭活或减毒的病原体,因为其成分比较复杂,并存在毒力返强等问题,所以其安全性一直让人担忧。而新型亚单位疫苗存在免疫原性弱、稳定性差、细胞微环境中易降解等问题,利用合适的递呈载体可以很好的解决这个问题。
鹅星状病毒是引起鹅痛风病的一个重要病原。鹅痛风病常在春、秋季暴发,临床表现为排泄白色或绿色稀便,关节出现肿大并伴有尿酸盐沉积和瘫痪。尸检时,在胆囊、关节剖面、输尿管和心、肝、气囊、气管、腺胃的表面以及胆汁内发现尿酸盐沉积,肾脏肿大发白。该病历时7-10d,发病率高达80-90%,若不及时治疗,死亡率可达20-70%。
目前雏鹅痛风病对鹅的养殖生产造成了巨大的影响,但尚无有效的商品化疫苗预防鹅星状病毒的感染。
发明内容
基于背景技术存在的技术问题,本发明的目的是通过原核表达技术,表达鹅星状病毒的主要抗原决定簇蛋白——鹅星状病毒纤突蛋白,鹅星状病毒纤突蛋白能刺激机体产生具有中和活性的抗体,从而达到保护雏鹅免受鹅星状病毒感染,为预防雏鹅痛风病带来了新的方法。
由于脂质体作为一种新型疫苗佐剂,是由非免疫原性、无毒性和可生物降解的磷脂组成的能够包裹抗原的类脂小球,非常适合作为递呈亚单位疫苗的工具,具有安全、耐受、缓释抗原等优点,本发明利用基因工程方法表达具有免疫原性的鹅星状病毒囊膜蛋白(VP27蛋白),采用薄膜扩散法制备空白脂质体,利用超声法将VP27蛋白包被在脂质体双分子层中。具体如下:
一种鹅星状病毒纤突蛋白脂质体疫苗,包括:脂质体,以及结合或包裹于脂质体中的VP27蛋白;
脂质体由卵磷脂、胆固醇、抗氧化剂按薄膜扩散法制得。
上述鹅星状病毒纤突蛋白脂质体疫苗制备方法,包括如下步骤:
S1、将卵磷脂、胆固醇、抗氧化剂混合,溶于有机溶剂中,减压旋蒸除去有机溶剂,得到膜状空白脂质体;
S2、向膜状空白脂质体中加入磷酸盐缓冲液和玻璃珠,水平摇床进行水化得到混悬液;
S3、将混悬液分离,加入VP27蛋白溶液,超声处理得到鹅星状病毒纤突蛋白脂质体疫苗。
优选地,S1中,卵磷脂为大豆卵磷脂、蛋黄卵磷脂、二油酰基卵磷脂、二油酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱中至少一种,抗氧化剂为维生素E、维生素A、维生素C中至少一种,有机溶剂为乙醇、乙醚、氯仿中至少一种。
优选地,S1中,卵磷脂、胆固醇、抗氧化剂的质量比为1.5-2.5:0.8-1.2:0.06-0.14。
优选地,S2中,磷酸盐缓冲液的pH值为7.3-7.5。
优选地,S2的水化过程中,水化温度为36-38℃,摇床转速为180-220r/min,水化时间为0.8-1.2h。
优选地,S3的超声处理过程中,超声功率为950-1050w,其中每工作5s间歇5s为1个周期,每60个超声处理周期后停止1min,超声处理总时间为32min。
优选地,S3的超声处理过程中保持冰浴,超声结束后除菌过滤。
优选地,S3中,VP27蛋白溶液采用如下工艺制备:将鹅星状病毒接种于病毒培养基中培养48-72h,提取RNA,反转录,PCR扩增获得VP27基因;将VP27基因和pCold质粒分别双酶切后连接,转化至Rosetta中,低温诱导表达,纯化得到VP27蛋白溶液。
优选地,鹅星状病毒源自安徽农业大学病原微生物与免疫学研究实验室分离保存的鹅星状病毒DY-19株。
优选地,S3中,加入VP27蛋白溶液后,向体系中加入蛋白酶抑制剂,然后进行超声处理。
优选地,蛋白酶抑制剂为抑肽酶、亮抑蛋白酶肽、EDTA中至少一种,蛋白酶抑制剂在体系中浓度为1.5-2.5μg/mL。
上述鹅星状病毒纤突蛋白脂质体疫苗在制备预防或治疗雏鹅痛风病的药物中的应用。
本发明的有益效果为:本发明通过薄膜扩散法,将具有免疫原性的鹅星状病毒纤突蛋白VP27包裹于脂质体中,延长抗原蛋白在机体内的作用时间,使免疫后动物机体能持续产生具有中和活性的抗体。
附图说明
图1为实施例1中抗原蛋白VP27的原核表达及其纯化的流程图。
图2为实施例1所得纯化后VP27蛋白的免疫印迹法验证图,其中M为蛋白分子量Marker,1为阴性对照,2为纯化VP27重组蛋白。
图3为实施例4所得脂质体疫苗负染后在透射电镜下的形态。
图4为实施例7中小鼠免疫后血清抗体时间-水平关系图。
具体实施方式
下面,通过具体实施例对本发明的技术方案进行详细说明。
实施例1抗原蛋白VP27的原核表达及其纯化
抗原蛋白VP27的原核表达及其纯化的流程如图1所示。选择源自安徽农业大学病原微生物与免疫学研究实验室分离保存的鹅星状病毒DY-19株,接种于LMH细胞上培养至F3代,收集细胞沉淀,通过Trizol法提取鹅星状病毒的RNA,使用Beyotime的M-MLV反转录试剂盒将提取的RNA反转录为cDNA,于-20℃保存备用。
将鹅星状病毒DY-19株于根据GenBank上的一株鹅星状病毒(登录号为:MG882765.1)设计一对特异性引物用于扩增VP27基因,具体如下:
GAstV-F:5'-CGGGATCCCAGGTTACTCCCTCGCTTGTG-3',
GAstV-R:5'-CCCAAGCTTAGAGGTCTTGAGCGAGACTGCTA-3',
基因两端分别设计了限制性内切酶BamHⅠ和HindⅢ位点,利用以上两个限制酶酶切位点将VP27基因与大肠杆菌表达载体pCold重组后转化大肠杆菌Rosetta菌株并表达,利用镍离子亲和层析柱完成纯化过程,纯化后VP27蛋白经免疫印迹法验证,如图2所示。图2证实本发明所得蛋白为46kDa,确为VP27蛋白。
实施例2制备空白脂质体
准确称取大豆卵磷脂1.13g、胆固醇0.57g、维生素E0.032g,溶解于20mL含有1%(V/V)乙醇的氯仿中,于37℃水平摇床中加速膜材的溶解。将溶解后的混合液加入250mL圆底烧瓶中,将烧瓶连接到旋转蒸发仪上。开启减压泵,42℃180r/min减压旋蒸除去有机溶剂,在瓶底形成一层均质薄膜,继续减压旋蒸30min除尽残留的溶剂,即得膜状空白脂质体。
实施例3水化
向上述圆底烧瓶中加入20mL pH值为7.4的磷酸盐缓冲液,同时加入小玻璃珠以加速水化过程,减小脂质体的粒径。置于水平摇床上,37℃200r/min水化1h,使脂质体从壁上完全溶解脱落,呈白色的悬浊液,将所得的悬浊液分离至烧杯中。
实施例4超声处理
向实施例1纯化所得的鹅星状病毒纤突蛋白(VP27蛋白)溶液中加入蛋白酶抑制剂,如抑肽酶、亮抑蛋白酶肽、EDTA等,本实施例选用抑肽酶,并使抑肽酶在鹅星状病毒纤突蛋白溶液中的浓度为2μg/mL。
取15mL含30μg抑肽酶的鹅星状病毒纤突蛋白溶液,加入实施例3所得悬浊液中,并在冰浴下进行超声处理。超声功率为1000w,其中每工作5s间歇5s为1个周期,每60个超声处理周期后停止1min,超声处理总时间为32min,最终得到均质乳白色混悬液,即脂质体疫苗。
将所得脂质体疫苗经滤器过滤后,装入灭菌的盐水瓶中,于4℃保存。
实施例5电镜观察
对实施例4所获得的鹅星状病毒纤突蛋白脂质体疫苗进行电镜观察,具体如下:
取20μL鹅星状病毒纤突蛋白脂质体疫苗样品进行负染,通过透射电镜观察脂质体疫苗的形态,如图3所示。
由图3可以发现:透射电镜下观察制得的脂质体都呈单室双层膜结构,形态均匀,表明此方法制得的脂质体形态良好。脂质体粒径决定了脂质体的大小形态稳定性,由此来判断是否适合用于注射。在《中国药典》对于混悬型注射液的药物粒度应控制在15μm以下,本发明所制备的脂质体粒度均控制在10μm以下,从而保证了脂质体的稳定性和适用性。
实施例6脂质体包封率测定
即脂质体包裹起来的抗原蛋白量与抗原蛋白总量的比例,是脂质体剂型必须评价的指标之一。
由于药物与脂质体结合的形式有三种:被包裹在脂质体颗粒内的水相、镶嵌在膜中、吸附在膜上。测定包封率需要将脂质体内包封的抗原蛋白与游离的抗原蛋白分离,然后进行相应的定量测定。两种组分的分离,一般采用离心法、半透膜分离法、凝胶过滤法等。
本实施例采用鱼精蛋白沉降法来测定脂质体包封率,实施例4所得疫苗在保证抗原蛋白活性的前提下,其包封率可达62%。
实施例7免疫原性
免疫原性反映了疫苗接种后引起动物机体产生免疫应答的强度和持续的时间,而免疫原性强弱取决于机体自身以及疫苗的质量,免疫原性是检测疫苗的有效性的重要指标之一。因此,本发明通过检测疫苗接种后机体产生抗体的水平,检验实施本发明所获得脂质体疫苗的免疫效果,即:采用实施例4所得疫苗免疫小鼠后,剪尾采集小鼠血清,通过间接ELISA检测小鼠血清特异性抗体的水平。具体如下:
将实施例中制得的鹅星状病毒纤突蛋白脂质体疫苗接种于6只6周龄、体重为28-30g的ICR小鼠,每只小鼠皮下接种0.2mL,首免后第二周进行加强免疫;
同时将VP27蛋白与完全弗氏佐剂另混合后,接种于6只6周龄、体重为28-30g的ICR小鼠,每只小鼠皮下接种0.2mL,首免后第二周用不完全弗氏佐剂与VP27蛋白混合进行加强免疫;
另取对照组6只昆明小鼠,皮下接种等量灭菌生理盐水,置于相同条件下饲养,首免后每周采集血清,用于后续抗体检测。
以上三组小鼠免疫前都经过采血,ELISA检测鉴定为阴性;其结果如图4所示。由图4可知:采用本发明所得脂质体疫苗接种后小鼠的血清抗体水平优于其余两组,证实本发明所得脂质体疫苗免疫原性强,使机体能持续产生具有中和活性的抗体。
以上所述,仅为本发明较佳的具体实施方式,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,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (9)
1.一种鹅星状病毒纤突蛋白脂质体疫苗,其特征在于,包括:脂质体,以及结合或包裹于脂质体中的VP27蛋白;
脂质体由卵磷脂、胆固醇、抗氧化剂按薄膜扩散法制得,其中抗氧化剂为维生素E、维生素A、维生素C中至少一种。
2.一种如权利要求1所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,包括如下步骤:
S1、将卵磷脂、胆固醇、抗氧化剂混合,溶于有机溶剂中,减压旋蒸除去有机溶剂,得到膜状空白脂质体,其中有机溶剂为乙醇、乙醚、氯仿中至少一种;
S2、向膜状空白脂质体中加入pH值为7.3-7.5的磷酸盐缓冲液和玻璃珠,水平摇床进行水化得到混悬液;
S3、将混悬液分离,加入VP27蛋白溶液,超声处理得到鹅星状病毒纤突蛋白脂质体疫苗。
3.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S1中,卵磷脂为大豆卵磷脂、蛋黄卵磷脂、二油酰基卵磷脂、二油酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱中至少一种;
S1中,卵磷脂、胆固醇、抗氧化剂的质量比为1.5-2.5:0.8-1.2:0.06-0.14。
4.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S2的水化过程中,水化温度为36-38℃,摇床转速为180-220r/min,水化时间为0.8-1.2h。
5.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S3的超声处理过程中,超声功率为950-1050w,其中每工作5s间歇5s为1个周期,每60个超声处理周期后停止1min,超声处理总时间为32min。
6.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S3的超声处理过程中保持冰浴,超声结束后除菌过滤。
7.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S3中,VP27蛋白溶液采用如下工艺制备:将鹅星状病毒接种于病毒培养基中培养48-72h,提取RNA,反转录,PCR扩增获得VP27基因;将VP27基因和pCold质粒分别双酶切后连接,转化至大肠杆菌Rosetta中,低温诱导表达,纯化得到VP27蛋白溶液。
8.根据权利要求2所述鹅星状病毒纤突蛋白脂质体疫苗制备方法,其特征在于,S3中,加入VP27蛋白溶液后,向体系中加入蛋白酶抑制剂,然后进行超声处理;
蛋白酶抑制剂为抑肽酶、亮抑蛋白酶肽、EDTA中至少一种,蛋白酶抑制剂在体系中浓度为1.5-2.5μg/mL。
9.如权利要求1所述鹅星状病毒纤突蛋白脂质体疫苗在制备预防或治疗雏鹅痛风病的药物中的应用。
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