CN114591406B - 一种传染性法氏囊病病毒重组vp2蛋白及其在疫苗中的用途 - Google Patents
一种传染性法氏囊病病毒重组vp2蛋白及其在疫苗中的用途 Download PDFInfo
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Abstract
本发明提供了一种重组VP2蛋白以及编码该蛋白的基因,本发明重组VP2蛋白与佐剂乳化后,制备的疫苗安全性高,对法氏囊经典株和变异毒株均具有很好的免疫效果,且所需免疫剂量小,能有效预防法氏囊经典株、变异株对鸡群的感染,具有推广应用价值。
Description
技术领域
本发明属于生物医药领域,具体涉及一种传染性法氏囊病病毒重组VP2蛋白及其在疫苗中的用途。
背景技术
由传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)引起的雏鸡传染性法氏囊病((Infectious bursal disease,IBD)是一种急性,高度传染性的免疫抑制性疾病。主要侵害中枢免疫器官-法氏囊,可造成机体发生严重的免疫抑制,从而降低对多种疫苗的反应性,增强对其他疾病的易感性,从而对养禽业造成了巨大的经济损失。
疫苗接种是控制传染性法氏囊病的重要措施,通过接种减毒活疫苗或者灭活疫苗使禽类产生主动免疫,从而预防传染性法氏囊病。VP2蛋白位于病毒粒子的外表面,与VP3共同构成核衣壳的骨架,是IBDV重要的毒力蛋白。大量研究证明,VP2蛋白决定了细胞嗜性,是IBDV毒力的主要决定因素。除此之外,VP2也是IBDV主要结构蛋白,能诱导中和抗体的产生,是主要的保护性抗原。目前研发的针对IBDV的多种基因工程疫苗都以VP2作为主要抗原基因。
然而,尽管疫苗接种策略是有效的,但IBDV自出现以来发生抗原漂移和抗原变异导致了抗原的多样性,目前IBDV有两个主要的血清型,分别为血清I型和血清II型,研究人员对I型病毒抗原性的研究发现I型病毒存在不同的亚型,具体可分为超强毒株、经典毒株及变异毒株。其中变异毒株及超强毒株从上世纪80年代末开始出现,由于其抗原性和病原毒力发生较大变异。而市面上大多数商品化的IBDV活疫苗都是基于经典毒株研制的,因而对于变异株,原有的商业疫苗难以起到良好的免疫保护作用,给该病的有效防控造成很大困难。
可见,传统的疫苗已经不能完全抵抗不断演变的IBDV病毒,迫切需要研究与流行毒株相匹配的新型疫苗。因此,研制一种生产效率较高并且免疫效果好的针对变异株的IBDV亚单位灭活疫苗对于目前临床防控IBD具有重要意义。
发明内容
本发明的目的在于提供一种针对IBDV变异株的传染性法氏囊病病毒重组VP2蛋白、重组病毒及传染性法氏囊病疫苗。
本发明提供了一种传染性法氏囊病病毒重组VP2蛋白,它的氨基酸序列如SEQ IDNO.1所示。
本发明还提供了上述重组VP2蛋白在制备传染性法氏囊病病毒疫苗中的应用。
本发明提供了一种编码传染性法氏囊病病毒重组VP2蛋白的基因,它的核苷酸序列如SEQ ID NO.2所示。
本发明提供了一种重组载体,它包括SEQ ID NO.2所示的核苷酸序列。
本发明提供了一种重组病毒,它包括SEQ ID NO.2所示的核苷酸序列,优选地,所述重组病毒是杆状病毒。
本发明还提供了上述的基因、重组载体和/或重组病毒在制备传染性法氏囊病疫苗中的用途。
本发明还提供了一种传染性法氏囊病病毒疫苗,它含有传染性法氏囊病病毒重组VP2蛋白抗原,所述传染性法氏囊病病毒重组VP2蛋白的序列如SEQ ID NO.1所示。
进一步地,它还含有佐剂;优选地,所述佐剂为白油司本佐剂。
进一步地,它还含有灭活的重组病毒,所述重组病毒包括SEQ ID NO.2所示的核苷酸序列;优选地,所述重组病毒是重组杆状病毒。
本发明还提供了上述疫苗的制备方法,它包括如下步骤:
(1)重组病毒接种HighFive细胞,于第4~5日收获上清,加二乙烯亚胺在30~37℃灭活病毒24~96小时,再加入硫代硫酸钠终止灭活,加表面活性剂混匀得到水相;所述重组病毒包括SEQ ID NO.2所示的核苷酸序列;
(2)佐剂灭菌,作为油相;
(3)向油相中加入水相的同时用乳化仪乳化,加入完毕后继续乳化至形成乳化剂,即得;
优选地,步骤(1)所述重组病毒接种HighFive细胞MOI为0.1~1,于第5日收获上清;所述二乙烯亚胺浓度为0.2%w/w,所述硫代硫酸钠浓度为0.02%w/w;所述表面活性剂是吐温-80或吐温-85,所述灭活病毒是32℃灭活96小时;
和/或步骤(2)所述佐剂是白油司本佐剂,白油和司本的质量比为94:6;
和/或步骤(3)所述油相和水相的体积比为2:1;加入水相的同时用乳化仪乳化时间为1min,转速12000r/min;加入完毕后继续乳化的时间为5min;转速18000r/min。
本发明的有益效果:
本发明提供了一种重组VP2蛋白,与佐剂乳化后,制备的疫苗安全性高,对法氏囊经典株和变异毒株均具有很好的免疫效果,且所需免疫剂量小,能有效预防法氏囊经典株、变异株对鸡群的感染。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为IBDV遗传进化树。
图2为sf9细胞病变情况。
图3为琼脂糖扩散试验表达的VP2蛋白效价结果(A)实施例3;(B)对比例1。
图4为VP2蛋白SDS-PAGE电泳结果。
图5为上清中VP2蛋白浓度测试结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
发明人前期由发病白羽肉鸡群的法氏囊病料组织接种SPF鸡胚分离获得一株分离株,动物攻毒实验发现,该病毒能引起鸡的法氏囊严重萎缩。完成测序获得VP2基因的序列信息,将编码VP2蛋白的氨基酸序列与基因库参考序列进行比对,构建遗传进化树,如图1所示,确定分离的病毒为变异株传染性法氏囊病毒,命名为CDSJ-1株。
实施例1、本发明重组VP2蛋白序列的确定
根据CDSJ-1株VP2基因测序翻译得到VP2蛋白序列,如下所示(SEQ ID NO.1):
MTNLQDQTQQ IVPFIRSLLM PTTGPASIPD DTLEKHTLRS ETSTYNLTVG DTGSGLIVFFPGFPGSIVGA HYTLQSNGNY KFDQMLLTAQ NLPASYNYCR LVSRSLTVRS STLPGGVYAL NGTINAVTFQGSLSELTDVS YNGLMSATAN INDKIGNVLV GEGVTVLSLP TSYDLGYVRL GDPIPAVGLD PKMVATCDSSDRPRVYTITA ADNYQFSSQY KTGGVTITLF SANIDAITSL SVGGELVFKT SIQNLVLGAT IYLIGFDGTAVITRAVAANN GLTAGIDNLM PFNLVIPTSE ITQPITSIKL EIVTSKSDGQ AGEQMSWSAS GSLAVTIHGGNYPGALRPVT LVAYERVATG SVVTVAGVSN FELIPNPELA KNLVTEYGRF DPGAMNYTKL ILSERDRLGIKTVWPTREYTDFREYFMEVA DLNSPLKIAG A
实施例2、合成重组VP2蛋白序列的密码子优化基因序列
根据VP2蛋白序列并合成相应的密码子优化的基因序列,如下所示(SEQ IDNO.2):
ATGACCAATC TACAGGATCA AACACAGCAG ATCGTCCCTT TTATTCGGTC TTTATTAATGCCCACGACTG GTCCGGCTTC GATCCCTGAC GATACTCTGG AGAAACATAC CCTACGCAGC GAGACATCAACCTACAATTT AACGGTCGGC GACACAGGAT CTGGCCTAAT AGTGTTCTTT CCCGGCTTTC CTGGCTCTATAGTGGGAGCC CATTATACTC TGCAGAGTAA CGGTAACTAC AAATTTGATC AAATGCTCCT CACGGCCCAGAATCTGCCAG CTTCATACAA TTACTGCCGG CTAGTTAGCA GATCGCTAAC GGTAAGATCC TCTACTCTTCCAGGCGGTGT TTATGCTCTC AACGGAACAA TCAATGCAGT GACTTTCCAG GGATCATTGA GCGAATTGACAGATGTTTCGTATAACGGCC TTATGAGTGC TACGGCCAAC ATTAATGACA AGATTGGAAA TGTATTGGTAGGTGAAGGAG TAACTGTACT GTCCCTTCCC ACGAGTTATG ACCTCGGCTA CGTCAGGCTA GGTGATCCTATACCAGCGGT AGGATTGGAC CCCAAAATGG TTGCGACGTG TGATAGTTCG GACAGACCCC GTGTTTATACCATTACGGCA GCGGATAATT ACCAATTCTC CTCACAATAC AAAACTGGCG GAGTCACCAT CACCCTCTTCTCGGCTAACA TTGACGCGAT TACTTCTTTA AGTGTGGGCG GAGAATTGGT CTTTAAGACG TCCATTCAGAATTTAGTCCT TGGCGCGACA ATCTATCTTA TTGGGTTTGA CGGGACCGCT GTCATTACGC GAGCAGTTGCCGCTAACAAT GGTCTAACTG CTGGTATAGA TAATCTAATG CCGTTCAATT TAGTTATACC GACTAGTGAGATCACGCAAC CGATAACCTC AATCAAATTA GAGATAGTGA CATCCAAGAG CGATGGACAA GCTGGCGAACAAATGTCGTG GTCTGCCTCT GGTAGCCTGG CCGTTACGAT ACACGGGGGC AACTACCCAG GGGCCCTCCGACCTGTGACT CTAGTTGCGT ATGAGAGGGT GGCAACAGGA TCGGTCGTCA CAGTAGCCGG GGTGTCCAACTTCGAGTTGA TACCTAACCC TGAACTCGCAAAAAACCTCG TGACCGAGTA CGGTCGGTTC GATCCTGGAGCCATGAACTA CACAAAATTA ATTTTGAGCG AAAGGGACCG CCTGGGGATC AAAACTGTAT GGCCCACCCGCGAGTATACT GATTTTCGAG AATATTTCAT GGAGGTGGCG GACCTGAACT CCCCGCTAAA GATAGCTGGTGCGTAA
实施例3、本发明重组质粒、病毒与IBD疫苗的制备:
(1)PCR扩增IBDV VP2基因
PCR扩增实施例2的IBDV-VP2基因序列,1%琼脂糖电泳,通过胶回收试剂盒纯化DNA序列。扩增引物序列如下:
F(SEQ ID NO.3):
CACCATCGGGCGCGGATCCATGACCAATCTACAGGATCAAA
R(SEQ ID NO.4):
TAGTACTTCTCGACAAGCTTTTACGCACCAGCTATCTTTAG
加样体系(50μl):
PCR扩增程序:
②③进行28个循环。
PCR产物纯化回收:
制备1%琼脂糖凝胶(含染料),将PCR产物样品加入加样孔,120V恒压进行电泳15min;
将目的DNA条带在切胶仪上切下来,放入干净的EP管,根据天根生化科技的DNA凝胶回收剂盒纯化回收DNA片段;
(2)连接PCR产物与供体质粒载体
将原pFastBacDual质粒载体用HindⅢ与BamHⅠ酶切,酶切体系如下:
50μl酶切体系:
37℃酶切4小时,然后加入6X DNA上样缓冲液,进行1%凝胶电泳,120V恒压进行电泳15min,然后纯化回收DNA片段,方法同上。
通过EasyGeno快速重组克隆试剂盒连接,连接体系如下:
50℃温育18min,然后放置冰上,转化到大肠杆菌TOP10感受态细胞中。转化步骤如下:
取50μL大肠杆菌感受态细胞,加入适量质粒(体积不超过4μL)冰浴30min后,42℃热激90s,马上放回冰上,冰浴2.5min;加500μL LB培养基,于37℃摇床慢摇振荡培养60min;取80μL涂在含有氨苄青霉素(100μg/mL)的LB固体培养基上,37℃倒置培养过夜。
(3)筛选,测序确认
从培养皿上挑起若干个单菌落到5mL LB培养基中过夜培养,通过质粒提取试剂盒提取供体质粒,然后酶切进行鉴定,最后送测序公司测序确认序列正确。
(4)转到DH10Bac感受态细胞
将测序正确的表达供体质粒pFastIBDV-VP2转化到DH10Bac感受态细胞,通过蓝白斑筛选挑取白色菌斑并用PCR的方法鉴定阳性菌落,提取基因组,即为重组杆状病毒基因-杆粒。
(5)转染细胞进行病毒包装
使用转染试剂Cellfectin(Invitrogen)将重组杆粒转入sf9细胞中,获得P1重组杆状病毒,将收获的病毒接种sf9,96h后观察到sf9细胞出现明显改变,证明成功包装杆状病毒,命名为BacIBDV-VP2。sf9细胞病变如图2所示。
(6)取重组杆状病毒接种HighFive第5日收获的上清加入0.2%(w/w)的二乙烯亚胺(BEI)在32℃条件下灭活96小时,加入0.02%(w/w)硫代硫酸钠终止灭活,制得IBDV-VP2亚单位灭活疫苗。按照不同AGP效价(1:3、1:6、1:12)配制3批疫苗(表1),加入吐温-80(PBS稀释)混匀后制备为水相,油相为白油和司本(94:6比例混合,为白油司本佐剂)灭菌后使用。60ml油相放入烧杯中,使用乳化仪12000r/min开始慢慢加入水相30ml(1min内加入),水相加入后改为18000r/min乳化5min。各个样取12ml,3000r/min离心15min,无水相析出,同时放入37℃培养箱放置,放置2周无分层。
表1 VP2蛋白实验苗制备
对比例1、CDSJ-1株原始基因序列及重组杆状病毒构建
(1)经基因测序,CDSJ-1株原始基因序列如下(SEQ ID NO.5):
ATGACAAACCTGCAAGATCAAACCCAACAGATTGTTCCGTTCATACGGAGCCTTCTGATGCCAACAACCGGACCGGCATCCATCCCGGACGACACCCTGGAGAAACACACTCTCAGGTCAGAGACCTCGACCTACAATTTGACTGTGGGGGACACAGGGTCAGGGCTAATTGTCTTTTTCCCTGGCTTCCCTGGCTCAATTGTGGGTGCTCACTACACACTGCAGAGCAATGGGAACTACAAATTCGATCAGATGCTCCTGACGGCCCAGAACCTACCGGCCAGCTACAACTACTGCAGGCTAGTGAGTCGGAGTCTCACAGTAAGGTCAAGCACACTCCCTGGTGGCGTTTATGCGCTAAACGGCACCATAAACGCCGTGACCTTCCAAGGGAGCCTGAGTGAACTGACAGATGTTAGCTACAATGGGTTGATGTCTGCAACAGCCAACATCAACGATAAAATTGGGAACGTCCTGGTAGGAGAAGGGGTAACCGTTCTCAGCTTGCCCACATCATATGATCTCGGGTATGTGAGGCTTGGTGACCCCATACCTGCTGTAGGGCTCGACCCAAAAATGGTAGCAACATGTGACAGCAGTGACAGGCCCAGAGTCTACACCATAACTGCAGCCGATAATTACCAATTCTCATCACAGTACAAGACAGGTGGGGTGACAATCACACTGTTCTCAGCCAACATCGATGCCATCACTAGTCTCAGCGTTGGGGGGGAGCTTGTGTTCAAAACAAGCATTCAAAACCTTGTGCTGGGCGCCACCATCTACCTCATAGGCTTTGATGGGACTGCGGTAATCACCAGAGCTGTAGCTGCAAACAATGGGCTGACGGCCGGCATCGACAATCTCATGCCCTTCAACCTTGTGATCCCGACCAGCGAGATAACCCAGCCAATCACATCCATCAAACTGGAGATAGTGACTTCCAAAAGTGATGGCCAGGCAGGGGAACAGATGTCGTGGTCGGCAAGTGGGAGTCTAGCAGTGACGATCCATGGTGGCAACTACCCAGGGGCTCTCCGTCCCGTCACGCTAGTGGCCTACGAACGAGTGGCAACAGGATCTGTCGTAACGGTCGCCGGGGTGAGCAACTTCGAGCTGATCCCAAATCCTGAACTAGCAAAGAACCTGGTCACAGAATACGGCCGATTCGACCCAGGAGCTATGAACTACACAAAACTGATACTGAGTGAGAGGGACCGTCTTGGCATCAAGACCGTCTGGCCAACAAGGGAGTACACTGACTTTCGCGAGTACTTCATGGAGGTGGCCGACCTCAACTCTCCCCTGAAGATTGCAGGAGCA
构建杆状病毒的方法同实施例3。
以下通过实验例证明本发明的有益效果。
实验例1、抗原表达及鉴定
(1)琼脂糖扩散试验(AGP)鉴定
采用实施例3的重组杆状病毒,以HighFive细胞密度为2×106个细胞/毫升,MOI为0.3PFU/细胞,125ml摇瓶放置27℃(转速120rpm)环境中进行抗原表达。5日后细胞活率降至10%以下,收获上清,琼脂糖扩散试验表明表达的重组VP2蛋白AGP效价为1:128(图3A),与之对比的原始序列(对比例1)表达重组VP2蛋白AGP效价为1:32(图3B),证明本发明优化的基因序列抗原表达量显著提升,表达量非常理想。
(2)P2蛋白SDS-PAGE灰度定量
采用实施例3的重组杆状病毒,以不同MOI接种3日后,SDS-PAGE电泳图42KD附近目的条带明显(图4),说明重组VP2蛋白表达。将已知浓度BSA标准品进行系列稀释,与收获的上清液共同进行SDS-PAGE,电泳结束后用QuantityOne软件(version:4.6.2)进行灰度分析进行目的蛋白定量灰度定量。结果显示,上清中VP2蛋白浓度可达到320ug/ml(图5)。
实验例2、免疫检测检验
(1)抗体水平检测
取60只21日龄SPF鸡,随机分成3组,20只/组,分别免疫实施例3所配制3批不同AGP的疫苗,0.3ml/只,颈部皮下注射。另取10只作为对照组,免疫后7、14、21、28天采血分离血清,AGP检测抗体水平。各组平均抗体水平如表2所示。抗体产生期显示各组疫苗组免后14天血清琼扩效价均不低于1:8。
表2杆状病毒表达VP2免疫原性试验抗体水平
(2)攻毒保护试验
免疫组每组取10只鸡,每只鸡点眼攻毒10倍稀释的经典强毒(BC6/85株)0.2ml(含有100BID),剩余10只鸡每只鸡点眼攻毒10倍稀释的分离变异CDSJ-1毒株0.1ml(含有100BID),对照组取5只攻毒BC6/85毒株,5只攻毒CDSJ-1株,剩余5只作为空白对照。攻毒后观察96h后全部剖检,观察法氏囊病变情况。
表3杆状病毒表达VP2蛋白免疫原性试验攻毒保护结果
攻毒后CDSJ-1株对照组死亡3只(攻毒后第3天死亡1只,出现严重啄尾现象,第4天死亡2只,),其余未出现死亡;剖检后CDSJ-1株对组发病率为5/5,死亡率3/5,法氏囊均出现萎缩、变黄、有黏液,免疫组发病率均为0/10。BC6/85攻毒组,对照组发病率5/5,法氏囊出现萎缩、变黄、有黏液、内部有干酪样物质,免疫组发病率均为0/10,详见表3。
上述结果说明表达的VP2蛋白免疫原性良好,对法氏囊经典株和变异毒株均具有良好的保护效果,配制成1:3/1:6/1:12琼扩效价疫苗效检合格,保护率均达到100%。
综上,本发明提供了一种重组VP2蛋白,与佐剂乳化后,制备的疫苗安全性高,对法氏囊经典株和变异毒株均具有很好的免疫效果,且所需免疫剂量小,能有效预防法氏囊经典株、变异株对鸡群的感染。
SEQUENCE LISTING
<110> 成都史纪生物制药有限公司
<120> 一种传染性法氏囊病病毒重组VP2蛋白及其在疫苗中的用途
<130> GY768-2022P0114971CCR3
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 441
<212> PRT
<213> VP2
<400> 1
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg
1 5 10 15
Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr
20 25 30
Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr
35 40 45
Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro
50 55 60
Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr
65 70 75 80
Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr
85 90 95
Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr
100 105 110
Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr
115 120 125
Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu
130 135 140
Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val
145 150 155 160
Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly
165 170 175
Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Val Gly Leu Asp Pro Lys
180 185 190
Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile
195 200 205
Thr Ala Ala Asp Asn Tyr Gln Phe Ser Ser Gln Tyr Lys Thr Gly Gly
210 215 220
Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu
225 230 235 240
Ser Val Gly Gly Glu Leu Val Phe Lys Thr Ser Ile Gln Asn Leu Val
245 250 255
Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile
260 265 270
Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr Ala Gly Ile Asp Asn
275 280 285
Leu Met Pro Phe Asn Leu Val Ile Pro Thr Ser Glu Ile Thr Gln Pro
290 295 300
Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Asp Gly Gln
305 310 315 320
Ala Gly Glu Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr
325 330 335
Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
340 345 350
Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val
355 360 365
Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val
370 375 380
Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu
385 390 395 400
Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr
405 410 415
Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu
420 425 430
Asn Ser Pro Leu Lys Ile Ala Gly Ala
435 440
<210> 2
<211> 1326
<212> DNA
<213> VP2 gene
<400> 2
atgaccaatc tacaggatca aacacagcag atcgtccctt ttattcggtc tttattaatg 60
cccacgactg gtccggcttc gatccctgac gatactctgg agaaacatac cctacgcagc 120
gagacatcaa cctacaattt aacggtcggc gacacaggat ctggcctaat agtgttcttt 180
cccggctttc ctggctctat agtgggagcc cattatactc tgcagagtaa cggtaactac 240
aaatttgatc aaatgctcct cacggcccag aatctgccag cttcatacaa ttactgccgg 300
ctagttagca gatcgctaac ggtaagatcc tctactcttc caggcggtgt ttatgctctc 360
aacggaacaa tcaatgcagt gactttccag ggatcattga gcgaattgac agatgtttcg 420
tataacggcc ttatgagtgc tacggccaac attaatgaca agattggaaa tgtattggta 480
ggtgaaggag taactgtact gtcccttccc acgagttatg acctcggcta cgtcaggcta 540
ggtgatccta taccagcggt aggattggac cccaaaatgg ttgcgacgtg tgatagttcg 600
gacagacccc gtgtttatac cattacggca gcggataatt accaattctc ctcacaatac 660
aaaactggcg gagtcaccat caccctcttc tcggctaaca ttgacgcgat tacttcttta 720
agtgtgggcg gagaattggt ctttaagacg tccattcaga atttagtcct tggcgcgaca 780
atctatctta ttgggtttga cgggaccgct gtcattacgc gagcagttgc cgctaacaat 840
ggtctaactg ctggtataga taatctaatg ccgttcaatt tagttatacc gactagtgag 900
atcacgcaac cgataacctc aatcaaatta gagatagtga catccaagag cgatggacaa 960
gctggcgaac aaatgtcgtg gtctgcctct ggtagcctgg ccgttacgat acacgggggc 1020
aactacccag gggccctccg acctgtgact ctagttgcgt atgagagggt ggcaacagga 1080
tcggtcgtca cagtagccgg ggtgtccaac ttcgagttga tacctaaccc tgaactcgca 1140
aaaaacctcg tgaccgagta cggtcggttc gatcctggag ccatgaacta cacaaaatta 1200
attttgagcg aaagggaccg cctggggatc aaaactgtat ggcccacccg cgagtatact 1260
gattttcgag aatatttcat ggaggtggcg gacctgaact ccccgctaaa gatagctggt 1320
gcgtaa 1326
<210> 3
<211> 41
<212> DNA
<213> F
<400> 3
caccatcggg cgcggatcca tgaccaatct acaggatcaa a 41
<210> 4
<211> 41
<212> DNA
<213> R
<400> 4
caccatcggg cgcggatcca tgaccaatct acaggatcaa a 41
<210> 5
<211> 1323
<212> DNA
<213> CDSJ-1
<400> 5
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcatc catcccggac gacaccctgg agaaacacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggcttcc ctggctcaat tgtgggtgct cactacacac tgcagagcaa tgggaactac 240
aaattcgatc agatgctcct gacggcccag aacctaccgg ccagctacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtaaggtca agcacactcc ctggtggcgt ttatgcgcta 360
aacggcacca taaacgccgt gaccttccaa gggagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgata aaattgggaa cgtcctggta 480
ggagaagggg taaccgttct cagcttgccc acatcatatg atctcgggta tgtgaggctt 540
ggtgacccca tacctgctgt agggctcgac ccaaaaatgg tagcaacatg tgacagcagt 600
gacaggccca gagtctacac cataactgca gccgataatt accaattctc atcacagtac 660
aagacaggtg gggtgacaat cacactgttc tcagccaaca tcgatgccat cactagtctc 720
agcgttgggg gggagcttgt gttcaaaaca agcattcaaa accttgtgct gggcgccacc 780
atctacctca taggctttga tgggactgcg gtaatcacca gagctgtagc tgcaaacaat 840
gggctgacgg ccggcatcga caatctcatg cccttcaacc ttgtgatccc gaccagcgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cttccaaaag tgatggccag 960
gcaggggaac agatgtcgtg gtcggcaagt gggagtctag cagtgacgat ccatggtggc 1020
aactacccag gggctctccg tcccgtcacg ctagtggcct acgaacgagt ggcaacagga 1080
tctgtcgtaa cggtcgccgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg tcacagaata cggccgattc gacccaggag ctatgaacta cacaaaactg 1200
atactgagtg agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact 1260
gactttcgcg agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320
gca 1323
Claims (11)
1.一种编码传染性法氏囊病病毒重组VP2 蛋白的基因,其特征在于,它的核苷酸序列如SEQ ID NO.2 所示。
2.一种重组载体,其特征在于,它包括SEQ ID NO.2 所示的核苷酸序列。
3.一种重组病毒,其特征在于,它包括SEQ ID NO.2 所示的核苷酸序列。
4.如权利要求3 所述的重组病毒,其特征在于,所述重组病毒是杆状病毒。
5.权利要求1 所述的基因、权利要求2 所述的重组载体或权利要求3 或4 所述的重组病毒在制备传染性法氏囊病疫苗中的用途。
6.一种传染性法氏囊病病毒疫苗,其特征在于,它含有传染性法氏囊病病毒重组VP2蛋白抗原,所述传染性法氏囊病病毒重组VP2 蛋白的序列如SEQ ID NO.1 所示;
它还含有灭活的重组病毒,所述重组病毒包括SEQ ID NO.2 所示的核苷酸序列。
7.如权利要求6 所述的疫苗,其特征在于,它还含有佐剂。
8.如权利要求7 所述的疫苗,其特征在于,所述佐剂为白油司本佐剂。
9.如权利要求6 所述的疫苗,其特征在于,所述重组病毒是重组杆状病毒。
10.权利要求6 所述疫苗的制备方法,其特征在于,它包括如下步骤:
(1)重组病毒接种HighFive 细胞,于第4~5 日收获上清,加二乙烯亚胺在30~37℃灭活病毒24~96 小时,再加入硫代硫酸钠终止灭活,加表面活性剂混匀得到水相;
(2)佐剂灭菌,作为油相;
(3)向油相中加入水相的同时用乳化仪乳化,加入完毕后继续乳化至形成乳化剂,即得。
11.如权利要求10 所述的制备方法,其特征在于,步骤(1)所述重组病毒接种HighFive细胞MOI 为0.1~1,于第5 日收获上清;所述二乙烯亚胺浓度为0.2%w/w,所述硫代硫酸钠浓度为0.02%w/w;所述表面活性剂是吐温-80 或吐温-85,所述灭活病毒是32℃灭活96 小时;
和/或步骤(2)所述佐剂是白油司本佐剂,白油和司本的质量比为94:6;
和/或步骤(3)所述油相和水相的体积比为2:1;加入水相的同时用乳化仪乳化时间为1min,转速12000r/min;加入完毕后继续乳化的时间为5min;转速18000r/min。
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