CN113425839B - 猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法 - Google Patents
猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法 Download PDFInfo
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- CN113425839B CN113425839B CN202110628735.9A CN202110628735A CN113425839B CN 113425839 B CN113425839 B CN 113425839B CN 202110628735 A CN202110628735 A CN 202110628735A CN 113425839 B CN113425839 B CN 113425839B
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- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法，涉及生物技术领域。本发明提供的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，包括猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原，抗原之间不存在干扰，免疫效力高，能够达到预防和治疗猪伪狂犬病和猪支原体肺炎的混合感染或继发感染的效果，显著降低了成本，节省了人力。
Description
技术领域
本发明涉及生物技术领域，尤其是涉及一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法。
背景技术
伪狂犬病(Pseudorabies，PR)是由伪狂犬病病毒(Pseudorabies vires，PRV)引起的多种家畜(猪、牛、羊等)和野生动物以发热、奇痒(猪除外)及神经系统症状和脑脊髓炎为主要特征的一种重要传染病。猪支原体肺炎又称猪地方性流行肺炎(Swine enzootichyopneumoniae)，俗称猪喘气病，是由猪肺炎支原体(Mycoplasma hyopneumoniae)引起的一种慢性、接触性、呼吸道传染病。由于联合疫苗(疫苗组合物)，如二联疫苗或三联疫苗中，多种抗原相互间存在干扰或影响，导致目前市场上还未见有猪伪狂犬病、猪支原体肺炎二联疫苗的产品。因此，目前对于预防猪圆环病毒、猪肺炎支原体共感染或继发感染，只能使用猪伪狂犬病疫苗、猪支原体肺炎疫苗分别进行二次免疫来预防和控制，导致免疫程序复杂，免疫次数增多，免疫成本增加，猪只的免疫应激次数增多，影响猪只的生长性能；或是，混合注射这2种疫苗来预防和控制，导致缺少免疫效率，且疫苗混合过程中不能保证无菌，操作过程繁琐。
有鉴于此，特提出本发明。
发明内容
本发明的第一目的在于提供一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗，以解决上述问题中的至少一种。
本发明的第二目的在于提供一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法，该制备方法简单快捷。
第一方面，本申请提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗，所述疫苗包括猪伪狂犬病抗原和猪支原体肺炎抗原；
所述猪伪狂犬病抗原包括猪伪狂犬gB抗原和猪伪狂犬gD抗原；
所述猪支原体肺炎抗原包括猪肺炎支原体CJ株抗原。
作为进一步技术方案，所述猪伪狂犬gB抗原的含量为100～150μg/头份，优选为100μg/头份。
作为进一步技术方案，所述猪伪狂犬gD抗原的含量为100～150μg/头份，优选为100μg/头份。
作为进一步技术方案，所述猪肺炎支原体CJ株抗原含量为109.0～1010.0CCU/头份，优选为7×109.0CCU/头份。
作为进一步技术方案，所述疫苗还包括佐剂。
作为进一步技术方案，所述佐剂包括水包油型佐剂和氢氧化铝胶；
作为进一步技术方案，所述水包油型佐剂占疫苗总体积的12％-18％，优选为15％；
作为进一步技术方案，所述氢氧化铝胶占疫苗总体积的12％-18％，优选为15％。
作为进一步技术方案，所述疫苗的免疫剂量为1.5～2.5mL/头，优选为2mL/头。
第二方面，本申请提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法，将猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原配制成抗原溶液，然后与佐剂混合，制备得到疫苗；
抗原溶液与佐剂混合的体积比为3:1～3:2，优选为7:3。
与现有技术相比，本发明具有如下有益效果：
本发明提供的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，包括猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原，抗原之间不存在干扰，免疫效力高，能够达到预防和治疗猪伪狂犬病和猪支原体肺炎的混合感染或继发感染的效果，显著降低了成本，节省了人力。
具体实施方式
下面将结合实施方式和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本发明，而不应视为限制本发明的范围。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前提下所获得的所有其他实施例，都属于本发明保护的范围。未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
第一方面，本申请提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗，所述疫苗包括猪伪狂犬病抗原和猪支原体肺炎抗原；
所述猪伪狂犬病抗原包括猪伪狂犬gB抗原和猪伪狂犬gD抗原；
所述猪支原体肺炎抗原包括猪肺炎支原体CJ株抗原。
猪伪狂犬gB抗原的氨基酸序列(SEQ ID NO.1)如下：
MPAGGGLWRGPRGHRPGHHGGAGLGRLWPAPHHAAAARGAVALALLLLALAATPTCGAAAVTRAASASPAPGTGATPDGFSAEESLEEIDGAVSPGPSDAPDGEYGDLDARTAVRAAATERDRFYVCPPPSGSTVVRLEPEQACPEYSQGRNFTEGIAVLFKENIAPHKFKAHIYYKNVIVTTVWSGSTYAAITNRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTNDTYTKIGAAGFYHTGTSVNCIVEEVEARSVYPYDSFALSTGDIVYMSPFYGLREGAHGEHIGYAPGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSFRFTSRALGASFVSDVTQLDLQRVHLGDCVLREASEAIDAIYRRRYNNTHVLAGDKPEVYLARGGFVVAFRPLISNELAQLYARELERLGLAGVVGPASPAAARRARRSPGPAGTPEPPAVNGTGHLRITTGSAEFARLQFTYDHIQAHVNDMLSRIAAAWCELQNKDRTLWGEMSRLNPSAVATAALGQRVSARMLGDVMAISRCVEVRGGVYVQNSMRVPGERGTCYSRPLVTFEHNGTGVIEGQLGDDNELLISRDLIEPCTGNHRRYFKLGGGYVYYEDYSYVRMVEVPETISTRVTLNLTLLEDREFLPLEVYTREELADTGLLDYSEIQRRNQLHALKFYDIDRVVKVDHNVVLLRGIANFFQGLGDVGAAVGKVVLGATGAVISAVGGMVSFLSNPFGALAIGLLVLAGLVAAFLAYRHISRLRRNPMKALYPVTTKALKEDGVEEDDVDEAKLDQARDMIRYMSIVSALEQQEHKARKKNSGPALLASRVGAMATRRRHYQRLENEDPDAP(SEQ ID NO.1)。
猪伪狂犬gD抗原的氨基酸序列(SEQ ID NO.2)如下：
MLLAALLAALVARTTLGADVDAVPAPTFPPPAYPYTESWQLTLTTVPSPFVGPADVYHTRPLEDPCGVVALISDPQVDRLLNEAVAHRRPTYRAHVAWYRIADGCAHLLYFIEYADCDPRQIFGRCRRRTTPMWWTPSADYMFPTEDELGLLMVAPGRFNEGQYRRLVSVDGVNILTDFMVALPEGQECPFARVDQHRTYKFGACWSDDSFKRGVDVMRFLTPFYQQPPHREVVNYWYRKNGRTLPRAYAAATPYAIDPARPSAGSPRPRPRPRPRPRPKPEPAPATPAPPGRLPEPATRDHAAGGRPTPRPPRPETPHRPFAPPAVVPSGWPQPAEPFPPRTTAAPGVSRHRSVIVGTGTAMGALLVGVCVYIFFRLRGAKGYRLLGGPADADELKAQPGP(SEQ ID NO.2)。
所述猪肺炎支原体CJ株，分离筛选自新疆维吾尔自治区昌吉市，该菌株的分类命名为：猪肺炎支原体，拉丁文学名：Mycoplasma hyopneumoniae，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：中国微生物菌种保藏管理委员会普通微生物中心，保藏日期：2014年11月06日，保藏编号：CGMCC NO：9099。
本发明提供的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，包括猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原，抗原之间不存在干扰，免疫效力高，能够达到预防和治疗猪伪狂犬病和猪支原体肺炎的混合感染或继发感染的效果，显著降低了成本，节省了人力。
在一些优选的实施方式中，所述猪伪狂犬gB抗原的含量为100～150μg/头份，例如可以为，但不限于100μg/头份、110μg/头份、120μg/头份、130μg/头份、140μg/头份或150μg/头份，优选为100μg/头份。
在一些优选的实施方式中，所述猪伪狂犬gD抗原的含量为100～150μg/头份，例如可以为，但不限于100μg/头份、110μg/头份、120μg/头份、130μg/头份、140μg/头份或150μg/头份，优选为100μg/头份。
在一些优选的实施方式中，所述猪肺炎支原体CJ株抗原含量为109.0～1010.0CCU/头份，例如可以为，但不限于1×109.0CCU/头份、2×109.0CCU/头份、4×109.0CCU/头份、6×109.0CCU/头份、8×109.0CCU/头份或1×1010.0CCU/头份，优选为7×109.0CCU/头份。
在本发明中，通过对疫苗中猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原含量的进一步优化和调整，使得疫苗中各个抗原含量适当，更好的实现对抗原的免疫。
在一些优选的实施方式中，所述疫苗还包括佐剂。佐剂的添加有助于提高疫苗的稳定性，提高病原体感染的免疫效力。
在一些优选的实施方式中，所述佐剂包括水包油型佐剂和氢氧化铝胶。其中水包油型佐剂包括但不限于SEPPIC ISA 11R、SEPPIC ISA 15A、SEPPIC ISA 28或SEPPIC ISA28R。在本发明中，将水包油型佐剂和氢氧化铝胶配合使用制备得到的疫苗通针性良好，具有免疫应答迅速、免疫效果持久、对动物机体的刺激更小的特点。疫苗注射后，氢氧化铝胶佐剂会迅速启动免疫，水包油佐剂延长免疫时间。首先，佐剂水相部分氢氧化铝胶释放出的抗原，刺激产生坚强的体液免疫反应；然后，大油滴表面的抗原刺激产生细胞免疫反应；最后，大油滴释放出小油滴的抗原，刺激产生终身的复合免疫力。
在一些优选的实施方式中，所述水包油型佐剂占疫苗总体积的12％-18％，例如可以为，但不限于12％、13％、14％、15％、16％、17％或18％，优选为15％；
在一些优选的实施方式中，所述氢氧化铝胶占疫苗总体积的12％-18％，例如可以为，但不限于12％、13％、14％、15％、16％、17％或18％，优选为15％。
通过对水包油型佐剂和氢氧化铝胶用量的进一步优化和调整，提高疫苗的免疫效力。
在一些优选的实施方式中，所述疫苗的免疫剂量为1.5～2.5mL/头，例如可以为，但不限于1.5mL/头、1.7mL/头、1.9mL/头、2.1mL/头、2.3mL/头或2.5mL/头，优选为2mL/头。
第二方面，本申请提供了一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法，将猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原配制成抗原溶液，然后与佐剂混合，制备得到疫苗；
抗原溶液与佐剂混合的体积比为3:1～3:2，优选为7:3。
本发明的猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法简单快捷方便。
本发明的猪伪狂犬病、猪支原体肺炎二联灭活疫苗含有猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原，免疫效力高，能够用于预防和治疗猪伪狂犬病和猪支原体肺炎的混合感染或继发感染。
下面通过具体的实施例和对比例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。
需要说明的是，以下实施例中所采用的CHO细胞含有编码猪伪狂犬gB、gD抗原的基因，并且能够表达猪伪狂犬gB、gD抗原。
改良Friis液体培养基由基础培养基为PPLO肉汤粉、脑心浸液粉、去离子水和琼脂粉，辅助培养基为10×汉克氏平衡盐溶液、酵母浸出液、青霉素、酚红和猪血清制成。
实施例1猪伪狂犬病、猪支原体肺炎二联灭活疫苗制备
1繁殖培养猪伪狂犬gB、gD抗原，具体方法如下：
1.1细胞复苏
从液氮罐中取出冻存管，迅速置于37℃恒温水浴锅中，轻轻晃动，使其快速融化。将冻存管中细胞悬液吸至离心管中，1000rpm离心5min，弃去上清，加入适量新鲜培养基重悬细胞，放入37℃，5％CO2培养箱中培养。
1.2细胞传代
CHO细胞培养于贴壁培养基中，37℃，5％CO2培养箱中培养。细胞长至融合度达80％以上时，将培养基吸出，用PBS洗涤2遍，加入1mL 0.25％胰酶溶液消化，轻晃细胞瓶待胰酶浸润瓶壁，吸出部分胰酶，以残留的少量胰酶继续消化，并至于倒置显微镜下观察，待细胞变圆变亮后倾去剩余胰酶溶液，加入含血清的新鲜培养基终止消化，并用吸管轻吹瓶壁至细胞全部脱落，弃去部分细胞液，吸管轻微吹制成单个细胞悬液，再加入适量培养基，放入培养箱中继续培养。悬浮细胞则取处于生长对数期细胞，计数，观察细胞状态，按照所需浓度和体积进行计算后，用一定量新鲜培养基重选，吹打混匀后置于100rpm/min，37℃恒温摇床中培养。
1.3蛋白纯化
收集细胞培养液，4℃，8000g离心30min，取上清，过0.8um滤膜，通过镍亲和层析进行蛋白纯化。将含有目的蛋白的咪唑洗脱液倒入透析袋内，用1×PBS透析至少10倍，留样检测。采用BCA法测定蛋白浓度；采用HPLC法检测纯度，纯度均需达到90％以上。在生物安全柜中，过0.22um真空滤器，过滤好的蛋白溶液样品存放于-80℃冰箱。
2培养猪肺炎支原体CJ株抗原，具体方法如下：
2.1生产用种子制备
从菌种库中取出生产种子，用改良Friis液体培养基溶解，并按1︰10～1︰15继代于改良Friis液体培养基中，置36～38℃恒温振荡培养箱中，以50～75r/min振荡培养42～48小时，待菌液颜色发生改变，pH值降低至6.70～6.80时收获菌液，作为一级种子，按现行《中国兽药典》附录进行纯粹检验，应纯粹。在2～8℃保存，使用期应不超过14日。继代应不超过5代。
2.2制苗用菌液制备
制苗用培养基为改良Friis液体培养基。将种子液按1︰10～1︰15加入改良Friis液体培养基中，密闭发酵罐罐口，通入无菌空气，罐内压力达到0.08MPa时，关闭发酵罐上所有进出口，保压，以50～100r/min搅拌，36～38℃培养42～48小时，pH值降低至6.70～6.80时取样，进行纯粹检验和活菌计数。
2.3灭活
制备0.1mol/L BEI溶液，过滤除菌，置2～8℃保存备用。将0.1mol/L BEI溶液加入菌液，终浓度为2mmol/L，37℃灭活24小时，以50～100r/min搅拌。灭活完成后加入1mol/L硫代硫酸钠，终浓度为20mmol/L，37℃反应1小时，置2～8℃保存，并进行灭活检验。
3疫苗制备
先将氢氧化铝胶佐剂和水包油型油佐剂分别除菌备用。将猪伪狂犬gB、gD抗原和猪肺炎支原体抗原按15︰15︰30比例(体积比)制成混合抗原，再将64.3％的混合抗原和氢氧化铝胶佐剂按45︰15比例低速搅拌混合，将35.7％的混合抗原和油佐剂按25︰15比例低速搅拌混合，最后将以上2种混合好的溶液低速搅拌混合。无菌定量分装，加盖密封。
实施例2不同抗原含量猪伪狂犬病、猪支原体肺炎二联灭活疫苗免疫效果验证
1材料
按照实施例1的方法制备得到如下三种疫苗。
疫苗1猪伪狂犬gB、gD抗原含量各为50μg/头份；猪肺炎支原体抗原含量为4×109.0CCU/头份，免疫剂量为2mL。
疫苗2猪伪狂犬gB、gD抗原含量各为100μg/头份；猪肺炎支原体抗原含量为7×109.0CCU/头份，免疫剂量为2mL。
疫苗3猪伪狂犬gB、gD抗原含量各为150μg/头份；猪肺炎支原体抗原含量为10×109.0CCU/头份，免疫剂量为2mL。
2方法
2.1安全性试验
用2～3周龄健康易感仔猪15头，每头耳后颈部深层肌肉注射2倍剂量(4mL)的免疫疫苗1、疫苗2、疫苗3，连续观察14日，未出现体温、食欲、精神状态，临床反应等的不正常情况和死亡情况。具体情况详见表1。
表1疫苗安全性试验情况
| 分组 | 体温 | 食欲 | 精神状态 | 临床反应 | 死亡 |
| 疫苗1 | 正常 | 正常 | 正常 | 无 | 无 |
| 疫苗2 | 正常 | 正常 | 正常 | 无 | 无 |
| 疫苗3 | 正常 | 正常 | 正常 | 无 | 无 |
2.2有效性试验
2.2.1猪伪狂犬部分有效性试验
筛选3～4周龄健康易感仔猪20头(猪伪狂犬病毒抗原抗体阴性)，随机分成4组，每组5头。其中1～3组分别免疫3种疫苗各2mL，第4组不注射作为对照，在同等条件下隔离饲养。免疫后28日，连同对照猪，各滴鼻接种经典强毒(猪伪狂犬病毒SC株)，观察14日。对照猪应至少4头发病，免疫猪应至少4头保护。
2.2.2猪肺炎支原体部分有效性试验
筛选3～4周龄健康易感仔猪20头(猪肺炎支原体抗原抗体阴性)，随机分成4组，每组5头。其中1～3组分别免疫3种疫苗各2mL，第4组不注射作为对照，在同等条件下隔离饲养。免疫后28日，连同对照猪，各气管注射猪肺炎支原体CJ株组织毒5mL(含0.01g肺组织毒，肺组织毒是由猪肺炎支原体CJ株攻毒后病变的肺脏制备的肺组织毒)，连续观察28日，进行剖检，按28分评分法对每头猪的肺炎病变进行评分，计算肺炎病变减少率。
疫苗有效性试验具体情况详见表2。
表2疫苗有效性试验结果
注：“/”表示不适用。
通过表2的实验数据说明，制备的3种不同抗原含量的猪伪狂犬病、猪支原体肺炎二联灭活疫苗免疫仔猪后，接种当前经典强毒(猪伪狂犬病毒SC株)攻击，分别能为仔猪提供60％(3/5)、100％(5/5)、100％(5/5)保护，而对照仔猪攻毒后100％发病。结果显示，疫苗2有很好的免疫保护效果。
通过表2的实验数据说明，制备的3种不同抗原含量的猪伪狂犬病、猪支原体肺炎二联灭活疫苗免疫仔猪后，接种猪肺炎支原体CJ株组织毒攻击，分别能为仔猪提供56.06％、75.76％、84.85％保护。结果显示，疫苗2有很好的免疫保护效果。
综上所述，猪伪狂犬gB、gD抗原含量各为100μg/头份，猪肺炎支原体抗原含量为7×109.0CCU/头份的猪伪狂犬病、猪支原体肺炎二联灭活疫苗免疫效果最佳。
实施例3猪伪狂犬病、猪支原体肺炎二联灭活疫苗各组份抗原干扰试验
1材料
按照实施例1的方法制备得到如下2种疫苗。
疫苗4猪伪狂犬gB、gD抗原含量各为100μg/头份，免疫剂量为2mL。
疫苗5猪肺炎支原体抗原含量为7×109.0CCU/头份，免疫剂量为2mL。
2方法
2.1猪伪狂犬部分抗原干扰试验
筛选3～4周龄健康易感仔猪15头(猪伪狂犬病毒抗原抗体阴性)，随机分成3组，每组5头。其中1～2组分别免疫2种疫苗各2mL，第3组不注射作为对照，在同等条件下隔离饲养。免疫后28日，连同对照猪，各滴鼻接种经典强毒(猪伪狂犬病毒SC株)，观察14日。对照猪应至少4头发病，免疫猪应至少4头保护。
2.2猪肺炎支原体部分抗原干扰试验
筛选3～4周龄健康易感仔猪15头(猪肺炎支原体抗原抗体阴性)，随机分成3组，每组5头。其中1～2组分别免疫2种疫苗各2mL，第3组不注射作为对照，在同等条件下隔离饲养。免疫后28日，连同对照猪，各气管注射猪肺炎支原体CJ株组织毒5mL(含0.01g肺组织毒)，连续观察28日，进行剖检，按28分评分法对每头猪的肺炎病变进行评分，计算肺炎病变减少率。
抗原干扰试验具体情况详见表3、4。
表3猪伪狂犬部分抗原干扰试验结果
注：“/”表示不适用。
表4猪肺炎支原体部分抗原干扰试验结果
注：“/”表示不适用。
通过表3、4的实验数据说明，制备的猪伪狂犬病、猪支原体肺炎二联灭活疫苗和猪伪狂犬病灭活疫苗免疫仔猪后，接种当前经典强毒(猪伪狂犬病毒SC株)攻击，分别能为仔猪提供100％(5/5)、100％(5/5)保护，而对照仔猪攻毒后100％发病。结果显示，猪肺炎支原体抗原对猪伪狂犬病、猪支原体肺炎二联灭活疫苗的免疫保护无影响。
通过表3、4的实验数据说明，制备的猪伪狂犬病、猪支原体肺炎二联灭活疫苗和猪支原体肺炎灭活疫苗免疫仔猪后，接种猪肺炎支原体CJ株组织毒攻击，分别能为仔猪提供78.26％、79.71％保护。结果显示，猪伪狂犬病抗原对猪伪狂犬病、猪支原体肺炎二联灭活疫苗的免疫保护无影响。
综上所述，确定猪伪狂犬病、猪支原体肺炎二联灭活疫苗各组份抗原相互无干扰。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 天康制药（苏州）有限公司
<120> 猪伪狂犬病、猪支原体肺炎二联灭活疫苗及其制备方法
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Claims (14)
1.一种猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述疫苗包括猪伪狂犬病抗原和猪支原体肺炎抗原；
所述猪伪狂犬病抗原为猪伪狂犬gB抗原和猪伪狂犬gD抗原；
所述猪支原体肺炎抗原包括猪肺炎支原体CJ株抗原；
所述猪伪狂犬gB抗原的氨基酸序列如SEQ ID NO.1所示；
所述猪伪狂犬gD抗原的氨基酸序列如SEQ ID NO.2所示；
所述猪伪狂犬gB抗原的含量为100~150μg/头份；
所述猪伪狂犬gD抗原的含量为100~150μg/头份；
所述猪肺炎支原体CJ株抗原含量为109.0~1010.0CCU/头份。
2.根据权利要求1所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述猪伪狂犬gB抗原的含量为100μg/头份。
3.根据权利要求1所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述猪伪狂犬gD抗原的含量为100μg/头份。
4.根据权利要求1所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述猪肺炎支原体CJ株抗原含量为7×109.0CCU/头份。
5.根据权利要求1所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述疫苗还包括佐剂。
6.根据权利要求5所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述佐剂包括水包油型佐剂和氢氧化铝胶。
7.根据权利要求6所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述水包油型佐剂占疫苗总体积的12%-18%。
8.根据权利要求7所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述水包油型佐剂占疫苗总体积的15%。
9.根据权利要求6所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述氢氧化铝胶占疫苗总体积的12%-18%。
10.根据权利要求9所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述氢氧化铝胶占疫苗总体积的15%。
11.根据权利要求1所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述疫苗的免疫剂量为1.5~2.5mL/头。
12.根据权利要求11所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗，其特征在于，所述疫苗的免疫剂量为2mL/头。
13.权利要求1-12任一项所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法，其特征在于，将猪伪狂犬gB抗原、猪伪狂犬gD抗原和猪肺炎支原体CJ株抗原配制成抗原溶液，然后与佐剂混合，制备得到疫苗；
抗原溶液与佐剂混合的体积比为3:1~3:2。
14.根据权利要求13所述的猪伪狂犬病、猪支原体肺炎二联灭活疫苗的制备方法，其特征在于，抗原溶液与佐剂混合的体积比为7:3。
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