CN114470181B - 一种猪圆环病毒2型（pcv2）的复合水佐剂疫苗及其制备方法 - Google Patents
一种猪圆环病毒2型（pcv2）的复合水佐剂疫苗及其制备方法 Download PDFInfo
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- CN114470181B CN114470181B CN202210156327.2A CN202210156327A CN114470181B CN 114470181 B CN114470181 B CN 114470181B CN 202210156327 A CN202210156327 A CN 202210156327A CN 114470181 B CN114470181 B CN 114470181B
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Abstract
本发明涉及一种猪圆环病毒2型(PCV2)的复合水佐剂疫苗及其制备方法。具体的，所述疫苗包含：(1)复合水佐剂；(2)猪圆环病毒2型(PCV2)抗原；所述的复合水佐剂中包含：黄姜皂素含量为1～10mg/ml，三七总皂苷含量为1～10mg/ml，枸杞多糖含量为1～10mg/ml，PEG E 400以重量计为0.5～2％；余量为水。
Description
技术领域
本发明属于生物技术领域，具体的，涉及一种猪圆环病毒2型(PCV2)的复合水佐剂疫苗及其制备方法。
背景技术
猪圆环病毒病是由猪圆环病毒2型(PCV2)引起的在我国养猪业存在较为广泛的猪传染性疾病，其中断奶仔猪为易感猪群，临床症状表现多为断奶仔猪多系统衰竭综合征，给我国养猪行业带来不小的经济损失。
疫苗佐剂是一种与抗原同时使用，且自身没有免疫原性，但能够刺激机体的非特异性和特异性免疫能力，使免疫系统快速识别和呈递抗原的物质。目前兽用疫苗佐剂主要分为油包水佐剂、水包油佐剂、水包油包水佐剂、水佐剂以及铝盐佐剂等，中药成分佐剂如生物碱、多糖类、皂苷类和植物油等。没有任何一种佐剂能够完美适用于所有抗原，不同的佐剂要配合相应的抗原使用，达到预防疾病的效果。
猪圆环病毒2型(PCV2)疫苗一般在仔猪断奶后进入保育阶段进行免疫，传统油佐剂疫苗在免疫后，由于免疫刺激性不够和仔猪体内母源抗体的干扰，在免疫后的一段时间内抗体水平表现为先抑后扬，由相当一段时期是处于低水平抗体期，无法有效应对疾病感染。
水佐剂疫苗的使用让仔猪在保育阶段相比油佐剂更加安全高效，另外水佐剂的添加量较少，免疫刺激性相比油佐剂更小，可以更快地进入育肥阶段，减少了因疫苗免疫给养殖户带来的损失。其中所用的皂素和皂苷以及多糖类物质皆为从中药类植物中提取提纯，代替了传统矿物油，解决其不可代谢、注射困难、注射部位炎症等问题，皂素和皂苷以及多糖类物质拥有抗病毒、抗感染、增强免疫力的作用，并且已被证实可通过调节动物的细胞免疫和体液免疫增强疫苗的免疫效果，当抗原进入仔猪体内，会迅速被免疫系统识别并引起一系列免疫反应，在短期内促进抗体的大量产生，使得保育期仔猪能够应对外源病毒的入侵。
发明内容
本发明首先涉及一种猪圆环病毒2型(PCV2)疫苗，所述疫苗包含：
(1)复合水佐剂；
(2)猪圆环病毒2型(PCV2)抗原；
所述的复合水佐剂中包含：黄姜皂素的添加量为1～10mg/ml，三七总皂苷的添加量为1～10mg/ml，枸杞多糖的添加量为1～10mg/ml，PEG E 400以重量计为0.5～2％；余量为水；
优选的，所述的复合水佐剂中包含：黄姜皂素的添加量为1mg/ml，三七总皂苷的添加量为5mg/ml，枸杞多糖的添加量为1mg/ml，PEG E 400以重量计为1％；余量为水；
所述的猪圆环病毒2型(PCV2)抗原为：圆环病毒cap蛋白，其氨基酸序列如SEQ IDNO.1所示。
SEQ ID NO.1：
MTYPRRRYRRRRHRPRSHLGQILRRRPWLLHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDGTIDYFQPNNRRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK
所述的抗原和水佐剂的比例为：25:1。
所述的复合水佐剂和所述的疫苗的PH值分别为5.8和6.5。
所述的黄姜皂素材料中，皂素含量大于90％；
三七总皂苷材料中：人参皂苷Rd总量大于80.0％；
枸杞多糖材料中，多糖含量大于50％。
本发明还涉及所述的猪圆环病毒2型(PCV2)疫苗的制备方法，所述方法包括如下步骤：
(1)将黄姜皂素、三七总皂苷、枸杞多糖加入水相溶解；
(2)100r/min转速下缓慢加入PEG E 400继续搅拌至无大颗粒不溶物后，将转速调至500r/min连续搅拌至泡沫消失得到复合水佐剂，水佐剂过滤除菌；
(3)按比例缓慢将水佐剂加入到水相抗原中，500r/min连续搅拌至澄清。
步骤(2)得到的复合水佐剂和步骤(3)得到的疫苗的PH分别为5.8和6.5。
本发明还涉及所述的猪圆环病毒2型(PCV2)疫苗在制备预防或治疗猪圆环病毒传染的制剂中的应用。
本发明的有益效果在于，
1、本发明涉及一种针对猪圆环病毒2型(PCV2)疫苗免疫的新型复合水佐剂，通过加入皂素和皂苷以及多糖类物质来提高机体非特异性免疫能力，增强猪圆环病毒2型(PCV2)抗原的免疫原性，
2、水佐剂不同于传统油佐剂，能够在疫苗注射后快速激活机体的体液免疫和细胞免疫，让免后的抗体产生速度快于传统油佐剂，并且免疫刺激性小，在免疫后不会对仔猪在保育过程中产生影响。
3、本发明作为一种针对猪圆环病毒2型疫苗(PCV2)复合中药提取物水佐剂，与目前业界大量使用的油佐剂相比，既能够快速刺激机体的体液和细胞免疫应答，还能使断奶仔猪迅速产生大量抗体以应对外界病毒感染，减小因母源抗体产生的免疫抑制。水佐剂疫苗易于注射，而且疫苗注射后，会被机体快速吸收，注射部位安全无残留。黄姜皂素、三七总皂苷、枸杞多糖作为中药提取物，免疫刺激性小，让免疫后的仔猪不会出现发烧和厌食等影响保育的症状，并极大地降低疫苗免疫的死淘率，减少养殖户的经济损失。
附图说明
图1、复合水佐剂疫苗对小鼠的免疫效力试验，图中显示的为复合水佐剂疫苗、ISA206佐剂疫苗、生理盐水抗体滴度对比图。
图2、复合水佐剂疫苗对仔猪的免疫效力试验，复合水佐剂疫苗、ISA206佐剂疫苗、生理盐水抗体滴度对比图。
具体实施方式
关键材料：
黄姜皂素(陕西亿昊川生物科技，HPLC检测含量：皂素95.0％)；
三七总皂苷(楚雄云植药业，HPLC检测含量：人参皂苷Rd总量为86.0％)；
枸杞多糖(陕西亿昊川生物科技，HPLC-UV检测含量：多糖≥50％)
实施例1、猪圆环病毒2型(PCV2)复合水佐剂疫苗配制方法的确定和制备
1、猪圆环病毒2型(PCV2)复合水佐剂疫苗配方确定
选取黄姜皂素、三七总皂苷、枸杞多糖作为水佐剂的主要成分，使用KollisolvPEG E 400(美国Sigma公司，货号:06855,CAS号：25322-68-3)作为非离子表面活性剂稳定水相。
设计正交试验，黄姜皂素、三七总皂苷、枸杞多糖分别设定三个浓度，PEG E 400与生理盐水配比量设定三组比例，按照正交试验原理分别配置成混合溶液，对其进行物理性状和稳定性指标检测，筛选出符合质量标准的疫苗复合水佐剂配方；将物理性状和稳定性合格的组注射6周龄BALB/c小鼠，观察各组小鼠的健康表现和存活状况，连续观察三天。
配制筛选结果为：黄姜皂素的添加量为1mg/ml，三七总皂苷的添加量为5mg/ml，枸杞多糖的添加量为1mg/ml，PEG E 400以重量计为1％。
表1、复合水佐剂各组分配比浓度设计
2.猪圆环病毒2型(PCV2)复合水佐剂疫苗制备
按照筛选结果量取一定量的黄姜皂素、三七总皂苷、枸杞多糖、PEG E 400，将黄姜皂素、三七总皂苷、枸杞多糖加入水相，
100r/min转速下缓慢加入PEG E 400继续搅拌至无大颗粒不溶物后，将转速调至500r/min连续搅拌10分钟后等待泡沫消失，
将配置好的复合水佐剂用0.22μm过滤器过滤除菌，然后按比例缓慢加入到水相抗原中，500r/min连续搅拌10分钟，猪圆环病毒2型(PCV2)复合水佐剂疫苗(最终抗原量和佐剂量的比是24:1)配置完成。
实施例2、猪圆环病毒2型(PCV2)复合水佐剂疫苗质量评价
1、检测离心后复合水佐剂疫苗的稳定性
10000r/min离心30分钟后仍维持均一、透明、澄清的外观，未出现有物质析出以及分层现象，与离心前对比颜色、澄清度、流动性均无变化，该猪圆环病毒2型(PCV2)复合水佐剂疫苗稳定性良好。
2、测定复合水佐剂和复合水佐剂疫苗的pH
各取一定量复合水佐剂和复合水佐剂疫苗至干净烧杯中，所使用的pH计品牌及型号为梅特勒-托利多(FE28)，分别测定三次，取平均值，复合水佐剂和复合水佐剂疫苗PH分别为5.8和6.5，在疫苗pH允许范围之内。
3、复合水佐剂疫苗稳定考察
各取三份疫苗分别于4℃、25℃放置6个月，37℃放置一个月，复合水佐剂疫苗外观均保持均一、透明、澄清的外观；10000r/min离心30分钟后仍维持均一、透明、澄清的外观，其稳定良好。
实施例3、猪圆环病毒2型(PCV2)复合水佐剂疫苗安全性试验
1、复合水佐剂疫苗对小鼠的安全性实验
20只体重18-22克的6周龄BALB/c小鼠，随机分成2组每组10只，分别皮下注射复合水佐剂疫苗500μl和生理盐水500μl，每日观察小鼠的健康表现和存活情况，连续观察7日，均未出现因疫苗注射而引起的不良反应和死亡。
2、复合水佐剂疫苗对仔猪的安全性实验
10只28-35日龄健康仔猪，经PCR和ELisa检测体内没有猪圆环病毒2型(PCV2)病毒和抗体，随机分成2组每组5只，检测并记录注射前2天的体温数据，每天检测2次；将2组仔猪分别颈部肌肉注射复合水佐剂疫苗4ml和生理盐水4ml，注射后每隔6小时测温，连续检测3天；观察仔猪的健康表现和存活状况，连续观察7日。其中疫苗组在注射疫苗后6小时体温较生理盐水组高出0.6℃，免后24小时逐渐恢复到免前体温水平；免后7天并未出现其他不良反应以及死亡情况。
实施例4、猪圆环病毒2型(PCV2)复合水佐剂疫苗免疫效力试验
1、复合水佐剂疫苗对小鼠的免疫效力试验
将复合水佐剂和猪圆环病毒2型(PCV2)抗原配制成疫苗，将ISA206佐剂(Seppic公司MONTANIDElMISA206VG)猪圆环病毒2型(PCV2)抗原配制成疫苗做为阳性对照。
30只体重18-22克的6周龄BALB/c小鼠，随机分成3组每组10只，分别皮下注射复合水佐剂疫苗100μl、ISA206佐剂疫苗100μl、生理盐水100μl，在免后7天、14天、21天、28天进行断尾采血，对血清进行间接ELISA实验，通过测定各组抗体滴度来判定各组抗体的消长情况，结果如图1所示，14-21天采血结果显示复合水佐剂组抗体提升明显，21-28天采血结果显示复合水佐剂组较ISA206佐剂组有显著优势。
2、复合水佐剂疫苗对仔猪的免疫效力试验
将复合水佐剂和猪圆环病毒2型(PCV2)抗原配制成疫苗，将ISA206佐剂猪圆环病毒2型(PCV2)抗原配制成疫苗。15只28-35日龄健康仔猪，经PCR和ELisa检测体内没有猪圆环病毒2型(PCV2)病毒和抗体，随机分成3组每组5只，分别皮下注射复合水佐剂疫苗2ml、ISA206佐剂疫苗2ml、生理盐水2ml，在免后14天、21天、28天、35天、42天、49天、56天、63天进行前腔静脉采血，对对血清进行间接ELISA实验，通过测定各组抗体滴度来判定各组抗体的消长情况，结果如图2所示，采血结果显示复合水佐剂组在免后14-21天抗体提升明显，在28天达到顶峰并在63天还未出现衰减趋势；而ISA206佐剂组在免后42天抗体才达到顶峰，且在63天出现抗体衰减趋势。
可见，本申请采用中药提取物黄姜皂素、三七总皂苷、枸杞多糖配合非离子表面活性剂的方法，成功制备出一种有效针对猪圆环病毒2型(PCV2)疫苗的免疫复合水佐剂。本申请作为水佐剂和猪圆环病毒2型(PCV2)抗原配制成疫苗后，相比传统油佐剂免疫刺激性更小，免疫效果明显高于传统油佐剂，随着免疫后时间的延续，这种效应更加明显，可以有效提高养殖户的经济效益。
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。
序列表
<110> 成都依思康生物科技有限公司
<120> 一种猪圆环病毒2型（PCV2）的复合水佐剂疫苗及其制备方法
<130> L-TN-CF220100S
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 234
<212> PRT
<213> Porcine circovirus
<400> 1
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Claims (6)
1.一种猪圆环病毒2型疫苗，所述疫苗包含：
(1)复合水佐剂；
(2)猪圆环病毒2型抗原；
所述的复合水佐剂中包含：黄姜皂素的添加量为1～10mg/ml，三七总皂苷的添加量为1～10mg/ml，枸杞多糖的添加量为1～10mg/ml，PEG E 400以重量计为0.5～2％；余量为水；
所述的抗原为猪圆环病毒2型cap蛋白；
所述的黄姜皂素中，皂素含量大于90％；
三七总皂苷中：人参皂苷Rd总量大于80.0％；
枸杞多糖中，多糖含量大于50％。
2.根据权利要求1所述的疫苗，其特征在于，所述的复合水佐剂中包含：黄姜皂素的添加量为1mg/ml，三七总皂苷的添加量为5mg/ml，枸杞多糖的添加量为1mg/ml，PEG E 400以重量计为1％；余量为水；
所述的猪圆环病毒2型cap蛋白的氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求1或2所述的疫苗，其特征在于，所述的复合水佐剂和所述的疫苗的pH值分别为5.8和6.5，所述的抗原和复合水佐剂的比例为：24:1。
4.权利要求1或2所述的猪圆环病毒2型疫苗的制备方法，所述方法包括如下步骤：
(1)将所述黄姜皂素、三七总皂苷、枸杞多糖加入水相溶解；
(2)100r/min转速下缓慢加入PEG E 400继续搅拌至无大颗粒不溶物后，将转速调至500r/min连续搅拌至泡沫消失得到复合水佐剂，水佐剂过滤除菌；
(3)按比例缓慢将水佐剂加入到水相抗原中，500r/min连续搅拌至澄清。
5.根据权利要求4所述的方法，其特征在于，步骤(2)得到的复合水佐剂和步骤(3)得到的疫苗的pH分别为5.8和6.5。
6.权利要求1或2所述的猪圆环病毒2型疫苗在制备预防猪圆环病毒2型传染的制剂中的应用。
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Address after: 610000 Chengdu Tianfu international biological city, high tech Zone, Chengdu, Sichuan (No. 618, Fenghuang Road, Shuangliu District) Applicant after: Chengdu yisikang Biotechnology Co.,Ltd. Applicant after: Hangzhou Yisikang Pharmaceutical Technology Co.,Ltd. Address before: 610000 Chengdu Tianfu international biological city, high tech Zone, Chengdu, Sichuan (No. 618, Fenghuang Road, Shuangliu District) Applicant before: Chengdu yisikang Biotechnology Co.,Ltd. Applicant before: Chengdu yisikang Pharmaceutical Technology Co.,Ltd. |
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