CN105002195B - 一种猪蓝耳病重组疫苗 - Google Patents
一种猪蓝耳病重组疫苗 Download PDFInfo
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- CN105002195B CN105002195B CN201510462709.8A CN201510462709A CN105002195B CN 105002195 B CN105002195 B CN 105002195B CN 201510462709 A CN201510462709 A CN 201510462709A CN 105002195 B CN105002195 B CN 105002195B
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Abstract
本发明公开了一种猪蓝耳病重组疫苗,将猪蓝耳病病毒ORF3基因和ORF5基因串联,构建了融合基因ORF3‑ORF5,插入真菌特异性双T‑DNA安全表达载体pCB130NG中,获得一种表达载体pCB130NG‑ORF3‑ORF5,利用PEG法、农杆菌介导法或电击法转化食药用真菌原生质体,经鉴定为阳性的菌丝体,培养获得T1代子实体,利用PCR筛选出只含目的基因而不含选择标记基因的子实体个体,培养至T2代,获得稳定表达猪蓝耳病病毒基因的转基因食药用真菌。通过转基因真菌菌丝的发酵培养,离心收集菌丝,烘干或冻干后,粉碎制备猪蓝耳病重组疫苗。利用本发明生产的猪蓝耳病疫苗各批次间质量差异小、产量大、成本低,且可以直接通过口服饲喂动物,具有广泛的社会效益和经济效益。
Description
技术领域
本发明属于基因工程和生物医药技术领域,具体涉及一种猪蓝耳病重组疫苗。
背景技术
猪蓝耳病又称“猪繁殖与呼吸综合症”,是由猪繁殖与呼吸综合症病毒(PRRSV)感染引起的以怀孕母猪繁殖障碍及各年龄段猪呼吸道疾病为主要特征的猪传染病。猪蓝耳病是一种在猪群中大范围流行的疾病,在许多国家都有发现且影响着全世界养猪业和食品的安全。因其是危害养猪业最严重传染病之一,现已被国际兽医局列为B类传染病,我国将其列为二类传染病。目前猪蓝耳病已遍布全球主要养猪国家和地区,给全球养猪业造成了巨大经济损失,对我国养猪业危害也极其严重。
传统疫苗的应用为我国动物疫病的防控做出了巨大贡献,目前依然是我国控制动物传染病的主要手段。目前针对高致病性猪蓝耳病的疫苗有灭活苗和活疫苗两种,2008年及以前,国内主要是采用灭活苗进行防疫,从2009年4月底,农业部开始批准了第一批共计4家企业生产高致病性蓝耳病活疫苗(JXA1-R株),活疫苗开始进入试点使用。两种疫苗各有优势,在免疫实践中,往往是针对不同的猪以及疫情情况使用不同的疫苗。根据农业部《2010年国家动物疫病强制免疫计划》,对商品猪和种母猪可选用灭活苗或活疫苗进行免疫,而种公猪选用灭活苗进行免疫。
但是,由于我国已经投入商品化的活疫苗毒株有很多种,造成我国猪场多种毒株同时存在的复杂情况,现有的疫苗鉴别诊断方法均无法从血清学上对疫苗免疫和野毒感染的猪进行区分,从而给疫病的防控和清除造成巨大的困难。而且目前我们仍未研究清楚能刺激产生保护性免疫的PRRSV抗原,不能有针对性的选取病毒免疫原来制备疫苗;而PRRSV是RNA病毒,病毒基因突变频率高,病毒蛋白抗原性多变,容易产生变异株逃逸免疫识别等诸多原因,传统的猪蓝耳病疫苗并不能达到很好的免疫效果。因此,研发一种能在血清学上区分疫苗免疫和野毒感染的新型安全高效的疫苗是必然趋势。
基因工程疫苗以其安全性好、质量均一、生产成本低、适合开发多价苗和联苗等诸多优点,成为新型疫苗的研究焦点和开发方向。猪蓝耳病病毒ORF5基因上包含T细胞、B细胞抗原决定簇,且其编码的GP5蛋白具有诱导产生中和抗体的能力,同时ORF5能引起细胞免疫应答。越来越多的证据表明ORF5基因是开发猪蓝耳病新型疫苗的首选靶基因。然而,目前许多实验结果表明利用ORF3基因编码的GP3蛋白开发的新型疫苗虽然检测不到抗猪蓝耳病病毒的中和抗体,但是对妊娠母猪的繁殖障碍可以提供部分保护。并且许多研究表明ORF5基因和ORF3基因串联的新型疫苗可以提供更好的免疫效果。但是,针对猪蓝耳病的有效基因工程疫苗还未上市,更不要说可饲的基因工程疫苗。因此,利用生物反应器开发可饲疫苗非常具有开发价值。
生物反应器是利用转基因技术或代谢调控技术在生物个体生产高附加值蛋白质和代谢产物,或者改造植物某种功能成分使之更适于人类利用的一种生物制药和蛋白生产技术。其中,被称为“天然生物反应器”的植物表达体系正在引起工业和医学界越来越多的关注,目前已成为生产高附加值的口服疫苗、医用蛋白、单克隆抗体的重要途径,是国外生物产业的研发热点之一。但目前动植物生物反应器仍然存在转基因困难、生长周期长、分离纯化困难、成本高及因担心基因漂移而造成的环境释放困难等问题。食药用真菌生物反应器作为动植物生物反应器的完美补充,具有基因转化容易、生长周期短、无需在室外环境中释放等优点,是具有重要开发价值的生物反应器受体系统。
发明内容
本发明的目的是提供一种融合基因pCB130NG-ORF3-ORF5及一种猪蓝耳病重组疫苗。
一种融合基因ORF3-ORF5,它的碱基序列如序列表SEQ ID NO.4所示。
一种融合蛋白ORF3-ORF5,它的氨基酸序列如序列表SEQ ID NO.5所示。
一种表达载体pCB130NG-ORF3-ORF5,它是:
1)用HindIII和EcoRI双酶切pCAMBIA1300载体,去除MCS区,经DNA聚合酶Klenow酶补平后,用T4连接酶连接形成中间载体pCB130-1;
2)在pCB130-1的SphI位点处引入SEQ ID NO.1所示的基因,包含左右边界序列和多克隆位点,构建成中间载体pCB130-2;
3)pCB130-2分别在PstI酶切位点处引入真菌特异性启动子GPD,在SacI与EcoRI酶切位点之间引入Nos终止子,构建成真菌特异性双T-DNA表达载体,命名为pCB130NG;
4)经Xbal和KpnI酶切,在pCB130NG中插入SEQ ID NO.4所示的基因。
一种猪蓝耳病重组疫苗,它是在食药用真菌的原生质中转染了一种表达载体pCB130NG-ORF3-ORF5,培养转基因菌丝;
所述的转基因菌丝经液体培养基发酵后,离心收集菌丝,烘干或冻干后,粉碎;
所述的食药用真菌为虫草、灵芝、金针菇或平菇。
本发明提供了一种猪蓝耳病重组疫苗,将猪蓝耳病病毒ORF3基因和ORF5基因串联,构建了融合基因ORF3-ORF5,插入真菌特异性双T-DNA安全表达载体pCB130NG中,获得一种表达载体pCB130NG-ORF3-ORF5,利用PEG法、农杆菌介导法或电击法转化食药用真菌原生质体,经鉴定为阳性的菌丝体,培养获得T1代子实体,利用PCR筛选出只含目的基因而不含选择标记基因的子实体个体,培养至T2代,获得稳定表达猪蓝耳病病毒基因的转基因食药用真菌。通过转基因真菌菌丝的发酵培养,离心收集菌丝,烘干或冻干后,粉碎制备猪蓝耳病重组疫苗。利用本发明生产的猪蓝耳病疫苗各批次间质量差异小、产量大、成本低,且可以直接通过口服饲喂动物,具有广泛的社会效益和经济效益。
附图说明
图1 真菌特异性双T-DNA安全表达载体pCB130NG示意图;
图2 pUC57-ORF3-ORF5载体酶切结果;
图3 pCB130NG-ORF3-ORF5转化后菌液PCR及质粒酶切结果;
图4 转基因蛹虫草菌丝的PCR检测;
图5 转基因蛹虫草的PCR检测;
图6 GP3、GP5特异性抗体水平检测结果;
图7 攻毒后小鼠直肠温度的测定;
图8 攻毒后小鼠各组织器官中病毒的积累。
具体实施方式
实施例1. 真菌特异性双T-DNA安全表达载体的构建
利用pCAMBIA1300载体作为基础载体,首先通过HindIII和EcoRI双酶切,去除MCS区,经DNA聚合酶Klenow酶补平后,用T4连接酶连接形成中间载体pCB130-1。接下来,通过基因合成技术在中间载体的SphI位点处引入一段序列(SEQ ID NO.1),包含左右边界序列和多克隆位点,构建成中间载体pCB130-2。在此载体基础上,通过PCR技术及酶切与连接方法,分别在PstI酶切位点处引入真菌特异性启动子GPD(SEQ ID NO.2),在SacI与EcoRI酶切位点之间引入Nos终止子(SEQ ID NO.3),构建成真菌特异性双T-DNA表达载体,命名为pCB130NG,该载体示意图见图1。
实施例2. 融合猪蓝耳病疫苗基因ORF3-ORF5的设计
为了提高GP3与GP5蛋白片段的免疫活性,将ORF3基因与ORF5基因串联一起进行构建,并且在两个蛋白片段中间插入LTB佐剂序列增强免疫效力;同时为使三个蛋白片段表达后在空间上隔开,在两个基因中间插入Linker;为便于表达后的检测,在ORF3基因的5’端和ORF5基因的3’末端加上His标签。得到的融合免疫蛋白GP3-GP5的氨基酸序列为SEQ IDNO.5序列。
实施例3. 融合基因ORF3-ORF5真菌表达载体的构建
猪蓝耳病病毒 ORF5 基因和ORF3基因为Genebank中已公布的基因序列(GenBank:ACF93752.1和ACF93750.1)。根据真菌对密码子的偏爱性对融合基因进行密码子优化后,通过人工合成方法合成SEQ ID NO.4,将该序列合成到载体pUC57上,并在序列两端添加酶切位点Xbal和KpnI。
将pUC57-ORF3-ORF5载体和本研究组改造后的真菌特异性表达双T-DNA安全表达载体pCB130NG(图1),分别用Xbal和KpnI酶切(图2),回收酶切产物后,经连接、转化、鉴定(图3),获得融合基因ORF3-ORF5真菌表达载体pCB130NG -ORF3-ORF5。
实施例4. 转ORF3-ORF5基因蛹虫草子实体的获得
1.蛹虫草原生质体的提取与转化:
(1)蛹虫草菌丝体的培养:将蛹虫草菌丝体于优化的固体PDA培养基培养9天,然后再接种于150mL液体PDA培养基,26-28℃、100-130r/min,250mL摇瓶振荡暗培养4天,直至长成生长量较多、大小均匀、状态良好的菌丝球。
(2)原生质体的制备:用0.8mol/L的甘露醇配制混合酶液,即溶壁酶和蜗牛酶复合添加,总量为3.0-4.0%,比例为1:1;按照菌丝体重量:酶液=1:6-10的比例酶解菌丝,酶解温度为34℃,酶解时间2.5-4小时;以无菌脱脂棉过滤除去残余蛹虫草菌丝体,5000g离心10min,收集原生质体。
(3)蛹虫草原生质体转化:将原生质体浓度调节到1-5×107个/ml,载体质粒的添加量为25-30µg,混合均匀,置于冰上5-10min后,加入30µl PEG 缓冲液(25% PEG4000,50mmol/L CaCl2,10mmol/L Tris-HCl,pH7.5),冰浴15-20min,再加入250µl PEG 缓冲液,室温放置20min,加入1ml的MYG培养液,4000g离心5min,弃掉上清液,用500µl MYG培养液回溶原生质体。
2.蛹虫草阳性转化菌丝的筛选和鉴定:
(1)蛹虫草阳性转化菌丝的筛选:经转化后的原生质体,在MYG培养液中培养5天后,均匀涂布于含有15-25mg/L潮霉素的MYG固体平板上,生长7天后,抗性转化子即可长出。继续培养抗性转化子,菌丝直径长到2cm左右时,将单个菌落接种到新的抗性PDA固体平板上(含有15-25mg/L潮霉素),培养至菌丝长满板。
(2)蛹虫草抗性转化菌丝的DNA提取:收集阳性转化菌丝,按照DNA提取试剂盒(爱思进生产)操作说明,提取抗性菌丝的基因组DNA。
(3)蛹虫草阳性转化子的鉴定:以抗性菌丝基因组DNA为模板,ORF3-ORF5基因5’端序列(ATGCATCATCACCACCACCAC)和3’端序列(TCAGTGGTGGTGATGATGATG)为引物,退火温度选择58℃,设计PCR反应。反应结束后,用琼脂糖凝胶电泳检测,扩增出目的基因条带的菌丝(图4),提取菌丝中蛋白,按照常规方法进行SDS-PAGE电泳,转膜后分别加一抗抗猪蓝耳病病毒的猪阳性血清和二抗HRP标记的兔抗猪IGg,,进行Western blot印迹分析,显示出GP3-GP5蛋白印迹的菌丝,即为转基因阳性转化子。
3.转基因蛹虫草子实体的筛选和获得:
(1)配制瓶料及接种:培养阳性转化菌丝作为菌种,配制蛹虫草培养料(大米45g,营养液45ml,营养液配方/L:蔗糖30g,蛋白胨150 g,硫酸镁0.5g ,磷酸二氢钾1.5g ,维生素B1 0.015 g,2,4-D 0.002 g),灭菌接种后,进行发菌和子实体培养管理。
(2)发菌:发菌温度控制在20-23℃,黑暗培养,持续15d左右,菌丝即可吃透培养料,完成营养生长。
(3)子实体培养:菌丝体成熟后,由白色逐渐转为桔黄色,此时,室内应增加光照,白天利用自然散射光,保持200勒克斯,晚间可利用日光灯作光源,每天应不少于10h光照,以促使菌丝体转色和刺激原基形成。待料面突起,并形成小米粒状原基时,要适当通风,补充新鲜空气,保护室内温度18℃-22℃,并提高空气相对温度至80%-85%。如湿度太大,易使培养基提早失水而影响产量。蛹虫草有较强的趋光性,因此在子实体形成后,应根据情况适当调整培养瓶与光源的相对方向,或调整室内光源方向,以保证子实体的正常生长形态,从而提高产量,形成橘黄色或橘红色的顶部略膨大的呈棒状的子座(子实体)。待子实体不再生长,其顶端出现许多小刺时,表明已成熟。(4)筛选:采取蛹虫草子实体提取基因组DNA,使用PCR技术筛选出只含有目的基因而无筛选标记的转基因蛹虫草(图5)。继续培养至T2代。
实施例5. 转基因蛹虫草菌丝的发酵培养
发酵培养获得含有重组融合免疫蛋白GP3-GP5菌丝:以筛选获得的转基因蛹虫草为母种,培养制得转基因蛹虫草菌丝作为发酵菌种,用PDA液体培养基为发酵液,按照菌种:发酵液=1:100的比例接种,发酵温度为28℃,大量制备蛹虫草菌丝体。离心收集蛹虫草菌丝体,离心力为1000g,将所获菌丝体低温烘干,即得到可饲的猪蓝耳病基因工程疫苗。
实施例6. 可饲猪蓝耳病基因工程疫苗的免疫活性检测
1.动物免疫:取8周大的母鼠随机分为空白对照组和免疫检测组,每组8只。免疫组小鼠饲喂转基因蛹虫草菌丝,空白组饲喂野生蛹虫草菌丝。在喂养的第1、15、30和45天给小鼠喂养一定剂量的蛹虫草菌丝后进行分开圈养。在小鼠初免前和每次免疫2周后进行采血检测。
2.血清中猪蓝耳病病毒GP3-GP5特异性抗体检测:以从病毒中纯化的GP3和GP5蛋白作为标准,以间接ELISA法测定血清抗体的OD值。以灭活猪蓝耳病病毒细胞毒为包被抗原,被检测小鼠血清为一抗,过氧化物酶标记的兔抗鼠IgG为二抗,在450nm处测定OD值,检测GP3-GP5蛋白在免疫小鼠血清中的表达水平(图6)。
3. 攻毒试验及临床症状观察:在小鼠第四次免疫后28天攻毒。对小鼠进行皮下刺穿攻毒,每只小鼠接种猪蓝耳病病毒强毒株。对攻毒后15天的小鼠的直肠温度(图7)以及身体各个组织器官内部病毒的积累等方面进行观察(图8)。
实施例7灵芝、金针菇或平菇转基因菌丝的制备
采用转基因蛹虫草菌丝相同的方法制备灵芝、金针菇或平菇转基因菌丝,按实施例6方法进行实验,均有较高的抗体水平,攻毒试验具有良好的保护作用。
<110> 吉林农业大学
<120> 一种猪蓝耳病重组疫苗
<160> 4
<210> 1
<211> 103
<212> DNA
<213> 人工
<400> 1
gtaaacctaa gagaaaagag cgtttaaagc ttctgcaggt cgactctaga ggatccccgg 60
gtaccgagct cgaattctgg caggatatat tgtggtgtaa aca 103
<210> 2
<211> 913
<212> DNA
<213> 人工
<400> 2
cgttcgtgac tcgcaatatc agtgcataat tgatgtctat caccgcagac acattcccta 60
cacgcaaccg catccctcga cattgaaccc cgtcgcatca tattctccgt cccttccatt 120
tacttcctcg atctcaatat agttctcgat gacgctgaat tcgttgccct caagggtctt 180
cgagccttcg acgtcgacaa tgcacgcgca gaatggagtg tgcgtaaaca tactttgact 240
atctactgtt aaccgctgta tggcttgaga cgtgtacaca ctgctgctct ctctcatatc 300
aaaggacttc tataaataga tctcaaccag tggcaaccgt tcttcaaatg tcatattttg 360
gtacgctgga gcagagtctc ttgtgcctgc actccgcacc accgacctcc tgatcgaatc 420
tcgagtacct cgatatcgag atggccgtgt gctacgacct tttccattac tccgtcaggc 480
tgtctacgat gtgtatcacg atatgggttg ggcctggggt tcaagttcca ctgttgaacc 540
agtgccccca tgttacctct ggaatcgtta tctcggtggt gtctgcgggg atatattcag 600
tgcgctaaaa acggaataca gctcgtctcc gtcttcctct cgggtcaaag ttacaaagaa 660
aggtggctaa atgtctggac atgaggtatt tgcatgacga aacgtctcca ctctcggagg 720
gttggcgcct aacaggatcg gctcacacaa ctgctgacaa gctagatctt gagtgagctt 780
ctaagctttt acacgacgat tacggcgagt cggtcctgtc cttctcatga agcgtgggac 840
tttcacgata tggcggtatt gatcatctaa tcacgccgtc ctatataaac ccctgcctcc 900
tctttcaccc gca 913
<210> 3
<211> 256
<212> DNA
<213> 人工
<400> 3
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atcggg 256
<210> 4
<211> 1830
<212> DNA
<213> 人工
<400> 4
atgcatcatc accatcacca cgccaactcg tgtacgttcc tccacatctt cctgcgctgc 60
ggttttctct actccttctg ttgcgccgtc gtcgccaact ccaacgccac cttctgcttc 120
tggtttcctc tcgtccgcgg caacttctcc ttcgagctga cggtcaacta taccgtctgt 180
cccccctgtc tcacccgtca agccgcggcg gaggtgtatg agcctggtcg ctcgctgtgg 240
tgccgtatcg gtcacgaccg ttgctccgag gaggaccacg acgagctcgg cttcatggtc 300
cctcccggcc tctcctcgga gggccacctc acgtccgtct acgcgtggct cgccttcctc 360
tccttttcgt acaccgcgca gttccatccc gagatcttcg gcatcggtaa cgtctcccgc 420
gtctacgtcg acatcaaaca tcaggtgatc tgcgcggtcc acgatggcga gaacgcgacc 480
ctcccccgtc acgacaacat ctcggccgtc taccagacct attaccagca ccaggtggat 540
ggcggcaact ggttccatct cgagtggctc cgccccttct tctcctcgtg gctggtcctc 600
aacgtctcgt ggttcctgcg tcgctcccct gcctcccacg tgtccgtccg tgtcttccgt 660
acctcccgcc ctacgctccc tcagcatcag gcgctcctgt cctcgacgac ctcggccgcg 720
ctcggcatgg ccacgcgtcc tctgcgccgt ttcgccaagg ccctctccgc cgcgcgtcgt 780
ggcggcggcg gctccggcgg cggcggctcc ggcggcggcg gctccatgaa caaggtgaag 840
tgctacgtcc tctttacggc cctgctctcc tcgctctacg cccacggtgc ccctcagacc 900
attaccgagc tctgttccga gtaccgcaac acccaaatct acaccattaa cgacaaaatc 960
ctctcgtaca ccgagtccat ggcgggcaag cgcgagatgg tcatcattac cttcaagtcg 1020
ggcgagacct ttcaggtgga ggtccccggc tcgcagcaca tcgactccca gaagaaggcc 1080
atcgagcgca tgaaggatac gctccgcatc acctacctca ccgagacgaa gattgacaag 1140
ctctgcgtct ggaacaacaa gacccctaat tccatcgccg cgatctccat gaagaacggc 1200
cccggcccca tgctcggcaa atacctcacg acgggctgtt gctcgcgcct cctctccctc 1260
tggtgcatcg tccctttctg cttcgccgtc ctggtgaacg cgaactcgaa ctcgtcctcc 1320
cagttccagc tcatctacaa cctgacgctc tgcgagctga atggcaccga ctggctggcc 1380
aacaagttcg actgggccgt cgaaacgttc gtcatcttcc ccgtcctcac ccatattgtc 1440
tcctacggcg cgctcaccac gtcgcatttc ctcgacacgg tgggcctcgt gacggtctcc 1500
accgccggct tctatcacgg ccgctatgtg ctgtcgtcga tctatgcggt ctgcgccctc 1560
gcggcgctca tctgcttcgt gatccgcctc gcgaagaact gcatgtcctg gcgttactcc 1620
tgcacccgct acaccaattt cctgcaagac acgaagggcc gcctgtaccg ctggcgttcg 1680
cccgtcatcg tcgagaaggg cggcaaggtc gaggtcgaag gccacctcat tgacctcaag 1740
cgtgtcgtcc tggatggctc cgtggcgacg cctctcaccc gtgtctccgc ggaacagtgg 1800
ggtcgcctcc atcatcatca ccaccactga 1830
<210> 1
<211> 609
<212> PRT
<213> 人工
<400> 1
Met His His His His His His Ala Asn Ser Cys Thr Phe Leu His Ile
5 10 15
Phe Leu Arg Cys Gly Phe Leu Tyr Ser Phe Cys Cys Ala Val Val Ala
20 25 30
Asn Ser Asn Ala Thr Phe Cys Phe Trp Phe Pro Leu Val Arg Gly Asn
35 40 45
Phe Ser Phe Glu Leu Thr Val Asn Tyr Thr Val Cys Pro Pro Cys Leu
50 55 60
Thr Arg Gln Ala Ala Ala Glu Val Tyr Glu Pro Gly Arg Ser Leu Trp
65 70 75 80
Cys Arg Ile Gly His Asp Arg Cys Ser Glu Glu Asp His Asp Glu Leu
85 90 95
Gly Phe Met Val Pro Pro Gly Leu Ser Ser Glu Gly His Leu Thr Ser
100 105 110
Val Tyr Ala Trp Leu Ala Phe Leu Ser Phe Ser Tyr Thr Ala Gln Phe
115 120 125
His Pro Glu Ile Phe Gly Ile Gly Asn Val Ser Arg Val Tyr Val Asp
130 135 140
Ile Lys His Gln Val Ile Cys Ala Val His Asp Gly Glu Asn Ala Thr
145 150 155 160
Leu Pro Arg His Asp Asn Ile Ser Ala Val Tyr Gln Thr Tyr Tyr Gln
165 170 175
His Gln Val Asp Gly Gly Asn Trp Phe His Leu Glu Trp Leu Arg Pro
180 185 190
Phe Phe Ser Ser Trp Leu Val Leu Asn Val Ser Trp Phe Leu Arg Arg
195 200 205
Ser Pro Ala Ser His Val Ser Val Arg Val Phe Arg Thr Ser Arg Pro
210 215 220
Thr Leu Pro Gln His Gln Ala Leu Leu Ser Ser Thr Thr Ser Ala Ala
225 230 235 240
Leu Gly Met Ala Thr Arg Pro Leu Arg Arg Phe Ala Lys Ala Leu Ser
245 250 255
Ala Ala Arg Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
260 265 270
Gly Gly Ser Met Asn Lys Val Lys Cys Tyr Val Leu Phe Thr Ala Leu
275 280 285
Leu Ser Ser Leu Tyr Ala His Gly Ala Pro Gln Thr Ile Thr Glu Leu
290 295 300
Cys Ser Glu Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile
305 310 315 320
Leu Ser Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile
325 330 335
Thr Phe Lys Ser Gly Glu Thr Phe Gln Val Glu Val Pro Gly Ser Gln
340 345 350
His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu
355 360 365
Arg Ile Thr Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp
370 375 380
Asn Asn Lys Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Lys Asn Gly
385 390 395 400
Pro Gly Pro Met Leu Gly Lys Tyr Leu Thr Thr Gly Cys Cys Ser Arg
405 410 415
Leu Leu Ser Leu Trp Cys Ile Val Pro Phe Cys Phe Ala Val Leu Val
420 425 430
Asn Ala Asn Ser Asn Ser Ser Ser Gln Phe Gln Leu Ile Tyr Asn Leu
435 440 445
Thr Leu Cys Glu Leu Asn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp
450 455 460
Trp Ala Val Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val
465 470 475 480
Ser Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu
485 490 495
Val Thr Val Ser Thr Ala Gly Phe Tyr His Gly Arg Tyr Val Leu Ser
500 505 510
Ser Ile Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile
515 520 525
Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr
530 535 540
Thr Asn Phe Leu Gln Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser
545 550 555 560
Pro Val Ile Val Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu
565 570 575
Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Leu
580 585 590
Thr Arg Val Ser Ala Glu Gln Trp Gly Arg Leu His His His His His
595 600 605
His
609
Claims (4)
1.一种表达载体pCB130NG-ORF3-ORF5,它是:
1)用HindIII和EcoRI双酶切pCAMBIA1300载体,去除MCS区,经DNA聚合酶Klenow酶补平后,用T4连接酶连接形成中间载体pCB130-1;
2)在pCB130-1的SphI位点处引入SEQ ID NO.1所示的基因,包含左右边界序列和多克隆位点,构建成中间载体pCB130-2;
3)pCB130-2分别在PstI酶切位点处引入真菌特异性启动子GPD,在SacI与EcoRI酶切位点之间引入Nos终止子,构建成真菌特异性双T-DNA表达载体,命名为pCB130NG;
4)经Xbal和KpnI酶切,在pCB130NG中插入SEQ ID NO.4所示的基因。
2.一种猪蓝耳病重组疫苗,它是在食药用真菌的原生质中转染了权利要求1所述的一种表达载体pCB130NG-ORF3-ORF5,培养转基因菌丝。
3.根据权利要求2所述的一种猪蓝耳病重组疫苗,其特征在于:所述的转基因菌丝经液体培养基发酵后,离心收集菌丝,烘干或冻干后,粉碎。
4.根据权利要求2或3所述的一种猪蓝耳病重组疫苗,其特征在于:所述的食药用真菌为虫草、灵芝、金针菇或平菇。
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