CN101024076A - 球虫的新用途 - Google Patents
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Abstract
本发明提供了一种球虫的新用途，具体地说是其作为疫苗活载体的应用。本发明进一步地提供了球虫载体活疫苗，其为能表达外源蛋白的转基因球虫或含有表达载体能表达外源蛋白的稳定转染球虫。本发明的球虫载体活疫苗可用于诱导机体同时产生保护性体液免疫和细胞免疫应答并产生记忆性应答，其实施简单，作用稳定，不产生免疫耐受，生物安全性高。
Description
技术领域
本发明涉及球虫的新用途，具体地说是球虫作为一种疫苗活载体的应用。
背景技术
利用转基因技术研制的以病原微生物为载体的重组疫苗既可避免弱毒疫苗株返强，又能产生细胞免疫应答，具有传统疫苗不可比拟的优点。目前活载体基因重组苗及其作用机制是国内外疫苗研究的重点领域。作为有效的疫苗活载体必须具备2个条件：其一是对宿主安全，其二是有效表达并运送有免疫活性的保护性抗原。
目前研究较多的疫苗载体有两大类：一类是弱毒病毒载体，如痘病毒和腺病毒；另一类是肠道病原菌弱毒菌，如沙门氏菌和乳酸杆菌。病毒活载体能通过将外源抗原展示在宿主细胞表面或以出芽方式释放到细胞外，被宿主免疫系统识别并引起有效的特异性免疫应答。遗传修饰后的弱毒肠道病原菌能在宿主体内持续高效地表达自身抗原和外源抗原，有效激发针对病原菌和外源抗原的保护性黏膜免疫、体液免疫和细胞免疫反应。致弱病毒和肠道病原菌作为活疫苗载体却有先天不足之处。作为疫苗载体，病毒和细菌可容纳的外源基因片段小，难以进行大规模的遗传修饰。病毒载体容易引起宿主免疫耐受，通过口服难以取得理想的免疫效果。肠道病原菌载体如沙门氏菌在宿主体内易扩散到全身其它部位，损伤其它器官和组织，并且持续感染引起免疫耐受；一些菌株属人兽共患病原，被排放到环境中后，对人类和动物的健康造成潜在威胁。而且细菌载体不能对表达的外源蛋白进行糖基化等修饰，表达的外源蛋白免疫活性不足。
相对于病毒和原核生物来说，利用真核生物作为载体无疑具有更大的优势。真核生物载体基因组更大，可以进行大规模的遗传修饰。而且真核细胞中蛋白可以进行正确折叠和修饰，表达的目的蛋白具有正常活性。尽管利用真核生物作载体面临的挑战远大于病毒和细菌载体，因为涉及到更为复杂的生物间相互作用，但在基因组和蛋白组研究已经相对成熟的今天来说，这种研究已经可以从设想变成现实。
以多种致弱的鸡球虫混合制成的活卵囊疫苗问世已经有十几年，目前其年销售额已超过3亿美元，在鸡球虫病的防治中发挥着越来越重要的作用。现在关于鸡球虫活卵囊疫苗与宿主间的相互作用以及宿主的抗球虫免疫应答机理的研究已经比较深入。然而，迄今还没有应用鸡球虫作为疫苗活载体的报道。
发明内容
本发明的目的在于提供一种球虫作为疫苗活载体的应用；
本发明的另一个目的在于提供一种球虫载体活疫苗。
通过本发明的球虫载体活疫苗表达目的蛋白，可以诱导机体同时产生保护性体液免疫和细胞免疫应答并产生记忆性应答。
将目的基因克隆到带有药物筛选标记的表达载体上，电穿孔法将重组载体导入球虫细胞内，药物筛选，获得整合表达目的蛋白的转基因球虫或含有表达载体能表达外源蛋白的稳定转染球虫。用转基因球虫或稳定转染球虫作为疫苗接种鸡后，球虫在发育过程中表达目的蛋白，蛋白表达的时间和空间受到球虫阶段性表达基因上下游序列的调控。
本发明的球虫载体可以用于表达巨型艾美耳球虫TFP250蛋白、禽流感病毒HA等纤突糖蛋白与其他外在和内在抗原蛋白(HA、NA、NP、M1、M2、NS1、NS2、PA、PB1和PB2蛋白)、鸡新城疫病毒F蛋白等病原体重要抗原蛋白，从而引起寄主的免疫应答。
本发明的疫苗活载体为早熟致弱或野生型鸡球虫，优选为柔嫩艾美耳球虫(Eimeria tenella)。
以柔嫩艾美耳球虫(Eimeria tenella)为代表的鸡球虫是进行转基因研究，进而作为活疫苗载体的较理想物种。由于球虫单倍体有14条染色体，基因组大小达到60Mb，因此有相当多的位点可用于整合，而且基因组可以允许大片段的外源基因插入。利用球虫自身的基因侧翼序列来调控外源保护性抗原基因的转录，使之在球虫的细胞内生长发育期阶段性地表达并分泌(利用球虫的信号肽)到虫体外，可获得稳定表达目的蛋白的激发宿主很强细胞免疫应答的转基因球虫。与细菌、病毒疫苗载体相比，转基因球虫作为活疫苗载体具有独特优势。第一，球虫核基因组大小约为60Mb，可以容纳大片段的外源基因。第二，球虫是真核生物，可以表达已糖基化修饰的活性抗原蛋白。第三，球虫进入肠上皮细胞后形成带虫空泡而与宿主细胞核隔离，因此其基因片段插入或整合到宿主细胞基因组中的概率很小。第四，由于卵囊壁的保护，转基因球虫活卵囊疫苗经口服后可以有效到达肠道的特定部位发挥疫苗作用，不会因为在嗉囊和胃等酸性环境降低疫苗的活性。第五，口服接种简单易行，适用于群体饮水免疫或拌料免疫；而且由于自然饲养条件下卵囊可以重复感染，免疫一次即相当于多次预防接种。第六，球虫的繁殖周期较短，且感染属于自限性，在生成卵囊以后即排到体外，不会引起免疫耐受问题。第七，球虫各个种均以鸡为惟一宿主，不会跨种间传播，而且其田间分布广泛，转基因球虫在田间的释放不会造成该种病原的散布，因而其生物安全性较高。
本发明疫苗活载体的建立标志着一种新型疫苗的诞生。本发明以活疫苗(活球虫卵囊)为载体表达另外一个或数个(类)疫苗用抗原，同时激发针对多种病原体的细胞免疫和体液免疫(包括粘膜免疫)，是对真核表达系统的有益补充；在球虫中表达生物体目的蛋白，为寄生虫学增添了新的研究方向；以本发明为基础研制的鸡球虫基因工程苗还可应用于生产，并完全可以有计划地控制我国球虫病的发生，使免疫鸡的成活率、增重及饲料报酬高于或等于使用抗球虫药鸡的生产性能，从而使广大养鸡户受益，且无公害无残留。
本发明有望在短期内取得具有源头创新性质的成果，实现我国在这一领域的跨越式发展，鸡球虫活载体疫苗的成功研制，将使我国在这一领域的研究居于世界前列，产品不仅可以保障国内养鸡业免受球虫病的困扰，而且还可以出口创汇，将成为兽医界科技产业可持续发展的生长点之一。
附图说明
图1是载体pHDEA-TFP构成模式图，图中H4：柔嫩艾美耳球虫组蛋白4上游调控序列；DHFR：弓形虫DHFR-TS基因编码区(针对乙胺嘧啶抗性)；EYFP2：增强型黄荧光蛋白基因编码区；Actin：柔嫩艾美耳球虫Actin基因下游调控序列。
其中，TFP为巨型艾美耳球虫TFP250基因，其他抗原蛋白基因插入位置相同。
具体实施方式
下面实施例用于对本发明的进一步说明，但不用来限制本发明的范围。
实施例1
本实施例拟以转基因柔嫩艾美耳球虫作为疫苗活载体，来表达目的基因，从而达到对球虫和某病原体的同时免疫。
以巨型艾美耳球虫TFP250基因为例：
1、构建表达载体以及克隆目的基因到表达载体上
将柔嫩艾美耳球虫BJ125株(索勋，曲鸿飞，刘宗权，林昆华，吕艳丽.柔嫩艾美耳球虫晚熟株选育及免疫原性研究.农业生物技术学报.1996，4(2)，78-79)接种10日龄鸡，5-7天后采集病鸡粪或肠道内容物获得球虫卵，孢子化后在载玻片上压出孢子囊，在显微镜观察下用毛细吸管将一个孢子囊吸上来，即为分离的柔嫩艾美耳球虫单孢子囊，分离的球虫单孢子囊经口感染鸡后收集增殖的卵囊，即获得纯化遗传相对一致的球虫虫株体系。获得遗传相对一致的柔嫩艾美耳球虫，纯化后，常规方法提取球虫基因组DNA，用PCR方法克隆球虫基因调控序列(组蛋白4上游调控序列H4)，RT-PCR方法获得巨型艾美耳球虫TFP250基因序列。将调控序列H4、药物筛选标记基因DHFR-TS、TFP250等元件构建到黄荧光载体(购自BD公司)上，构建柔嫩艾美耳球虫转基因所需的表达载体pHDEA-TFP，具体如下：
克隆弓形虫DHFR-TS基因编码区：提取弓形虫总RNA，用RT-PCR方法扩增获得DHFR-TS基因，两端分别引进SacII和BamHI酶切位点，设计上下游引物(DHFR-UP：5′-CAT
CCGCGGATGCAGAAACCGGTGTG-3′；DHFR-L：5′-CT
GGATCCAAGACAGCCATCTCCATC-3′)，获得大小为1.8Kb的DNA片段，回收目的片段，将其与载体PGEM-T连接，连接过夜后转化，提取质粒，酶切鉴定，对所得候选阳性质粒进行DNA测序后PCR法引入抗乙胺嘧啶突变位点。
克隆柔嫩艾美耳球虫组蛋白4上游调控序列和柔嫩艾美耳球虫Actin基因下游调控序列：以球虫基因组DNA为模板，在上游调控序列两端引进NdeI和SacII酶切位点(H4-UP：5′-CAG
CATATGAACCAGCAAAGGTAGCAAC-3′；H4-L：5′-CTACCGCGGGATACCCTGGATGTTGTC-3′)以扩增H4上游调控序列；在带有黄色荧光蛋白基因和下游调控序列两端引进BamHI和MfeI位点，分别设计引物(E2-UP：5’-CATCCAGGGTATCGGATCCTGTCG-3′；Actin-L：5′-CGCAATTGCTTCACATGGAACCCCTGG-3′)，用PCR方法扩增出黄色荧光蛋白基因和下游调控序列。分别获得了大小为2.0kb与2.9kb的上游调控序列及与黄色荧光蛋白融合的下游调控序列的DNA片段，回收目的片段，将其与载体PGEM-T连接，连接过夜后转化，提取质粒，酶切鉴定，对所得候选阳性质粒进行DNA测序。
克隆巨型艾美耳球虫TFP250基因：提取巨型艾美耳球虫总RNA并反转录获取cDNA，PCR扩增时在TFP250两端分别引进NotI和融合后的SacII和BamHI位点(T-UP：5’ATC
CCGCGGGCCCG
GG ATCCTGTCGCCACCATGGAATTGCACCCCATTCCAG 3’；T-L：5′ATTT
GCGGCCGCCTACTGAATGTCGCCGCTGTCG 3 ′)并扩增，获得了大小为660bp的DNA片段，回收目的片段，将其与载体PGEM-T(购自Promega公司)连接，连接过夜后转化，提取质粒，酶切鉴定，对所得候选阳性质粒进行DNA测序。
将各基因酶切后与相应酶切后的黄荧光载体连接，最终获得包含各元件的表达载体，其核苷酸序列如序列表SEQ ID NO.1所示。
2、转化球虫并筛选
将分离的柔嫩艾美耳球虫单卵囊经口感染鸡后收集增殖的卵囊，上述卵囊按每羽200-500个接种15日龄鸡，5-7天后收集纯化最早排出的球虫卵囊，如此进行连续传代，即为所需载体用卵囊。
通过电穿孔法将构建好的pHDEA-TFP转染到柔嫩艾美耳球虫子孢子内，再接种MDBK(购自欣经科公司)单层培养细胞进行体外培养，同时加特异性的乙胺嘧啶抗性筛选药物。具体为：取早熟株子孢子1×107个，加入pHDEA-TFP质粒50μg，电击条件为2kv，电容为25μF，电击时间为0.3ms，静止20min后将转染后的子孢子悬液加入到生长至80％满的MDBK单层细胞上，41℃培养。孵育24小时后施加选压力，即加入特异性的乙胺嘧啶抗性筛选药物，至荧光显微镜下观察。
乙胺嘧啶药物压力筛选下得到稳定表达DHFR-TS、YFP与目的蛋白TFP250的球虫，重复这一筛选过程即可获得稳定表达的转基因球虫系或稳定转染球虫系。荧光显微镜下可观察到稳定表达转基因球虫或稳定转染球虫系的虫体可发黄荧光，发光率高于99％。
3、接种鸡
先体外孢子化卵囊，选择1-5日龄健康鸡口服接种经选育的早熟株或野生型转基因球虫或稳定转染球虫孢子化卵囊，200-500个球虫/只，5-7天后检测临床表现、每克粪便中卵囊数(O.P.G)、病变计分等指标。
4、效果
接种后发现该转基因球虫或稳定转染球虫对柔嫩艾美耳球虫与巨型艾美耳球虫或其他病原的感染有良好的免疫原性，接种后病变记分下降，感染鸡的球虫卵囊产量降低，盲肠病变值降低，鸡的增重下降也得到改善，O.P.G值为6×104-6×106，对致死剂量的球虫攻击或其他病原攻毒感染产生良好的免疫保护。
序列表
<110>中国农业大学
<120>球虫的新用途
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>14355
<212>DNA
<213>人工序列
<400>1
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgaa 240
ccagcaaagg tagcaacagt agaggttagc aatgttacct ttgtcaccgt cagtgacagg 300
cacaagaacg aacttgttac actcgagata catagcttct gtaaaactga atcattcgcg 360
gtttctgttt gctctgctga catctattgg tccattgttg gagactctgt tgctttttct 420
gttttttaaa ttcttataga acccgctatt gctgtcgtgg tgagccatcc gccgcgaaac 480
ggctgatgct cgactcattg tggctgcttt gcccattcgc tgcggttagt tgcagaaaaa 540
aacagcggca caaatagctg ctcccccagg tgataatatc tctacaacag cattctagga 600
tgtgcttgaa tgtcccgccc tcttcctttc caaatagttg ctcgcgcagt tgatgatatc 660
gtaacagcag cgttctagtg agtatttgaa tacccagccc tgttgttttc aaagccacgc 720
aacagcagca gttacagaag catataaagc ccatgcagct gatgctgctg cagctgaact 780
ctctactggg aaaagcatgc atcatgcaaa atgctgtgca acctgcgggg caaagcatgc 840
aaaaagctcc cctctaaggg gatgatatgt gtgactctca ataggcgtac tggttttgct 900
gccagaagct tccgctagtg tgaccaatac atttagagtt tgccattacc actggtgtta 960
aggacctccg taaagagctt ttggaaggcg gcagttcgag caagcaggag ctggactgcc 1020
tttaacagcc attggcaagt gaaaccatcg aatcactgtt actcacacag acgggcattt 1080
gctcgcactt ttttggttag ggcctcaagg gaatttgggg cagcagtgga ttagcacctc 1140
cactgctctt ttcgccaatt cttccgtcac cccgggttta caaacgaacg agctcgtagc 1200
tgtagtcccc ggcgtttgta ctgtgccccg cagtaagtgg gacgcactaa ggtcctccta 1260
ctttaacagt tcgagttcct tcttctcgcg ctcggctttc cgctctcgca tgtatcttgc 1320
accactgcaa ctgcaacatt cagtgactta gccgtcagat ggcatctgca gtgctgttgt 1380
cattaataaa tttcatcgtt gctgtacata gcctgtttat cacgcgacta tattgctaaa 1440
cgatgcaaag acagaagtgc cagcagcagc agaagcagcc taaacggcgc aaaccctgtc 1500
gggtggcagc ccgttaattt cgaagcatag tcatgcgtgc ttgcagcact tcagacactc 1560
aaaaaaataa tgcatatctc cacacaaatg caggcatatt ctcaacgtct cagtaggttg 1620
ctatgacctg aactgtgggg ggtggttgtt atacagaagg cgacgtaaac gaagtcctgc 1680
ggggactgaa caataatcta cggatgtatt tagtcctaaa agcacaaact tgatggcagt 1740
tggcagaaga aacatttaaa gaattagttt taaccaaaag gctacgagcg agggaggagg 1800
cgcggggatg ctgcagcacc tcctctacca ccctgtgtac ttccgtacgt tgcacaggtg 1860
gcggtgaata acgtccgtcg gttctacttg catccttaag ctgttattaa agcgtaattt 1920
tgcaaaaaat taggatagag aataaaatat ttggaaaaca atggcgcttt atttgctgaa 1980
acaacaagcg aggctggggg tcggctgttc gcccgggcgc gggctagcag gctagctgcg 2040
cagagactgc atgttccccc actgtacgtg cagggtgcgc gtgttgttgt tcgtgatggt 2100
atcggcgcgc gtatgtctca catccgttat ttagaggcta caacgagact gtctgttgtg 2160
tctgttccat agaaaaccaa aatgtctggc cgaggaaaag gaggaaaggg gttgggaaag 2220
ggaggcgcca agcgccaccg caaggtgttg cgcgacaaca tccagggtat cccgcggatg 2280
cagaaaccgg tgtgtctggt cgtcgcgatg acccccaaga ggggcatcgg catcaacaac 2340
ggcctcccgt ggccccactt gaccacagat ttcaaacact ttcgtcgtgt gacaaaaacg 2400
acgcccgaag aagccagtcg cctgaacggg tggcttccca ggaaatttgc aaagacgggc 2460
gactctggac ttccctctcc atcagtcggc aagagattca acgccgttgt catgggacgg 2520
aaaaactggg aaagcatgcc tcgaaagttt agacccctcg tggacagatt gaacatcgtc 2580
gtttcctctt ccctcaaaga agaagacatt gcggcggaga agcctcaagc tgaaggccag 2640
cagcgcgtcc gagtctgtgc ttcactccca gcagctctca gccttctgga ggaagagtac 2700
aaggattctg tcgaccagat ttttgtcgtg ggaggagcgg gactgtacga ggcagcgctg 2760
tctctgggcg ttgcctctca cctgtacatc acgcgtgtag cccgcgagtt tccgtgcgac 2820
gttttcttcc ctgcgttccc cggagatgac attctttcaa acaaatcaac tgctgcgcag 2880
gctgcagctc ctgccgagtc tgtgttcgtt cccttttgtc cggagctcgg aagagagaag 2940
gacaatgaag cgacgtatcg acccatcttc atttccaaga ccttctcaga caacggggta 3000
ccctacgact ttgtggttct cgagaagaga aggaagactg acgacgcagc cactgcggaa 3060
ccgagcaacg caatgagctc cttgacgtcc acgagggaga caactcccgt gcacgggttg 3120
caggctcctt cttcggccgc agccattgcc ccggtgttgg cgtggatgga cgaagaagac 3180
cggaaaaaac gcgagcaaaa ggaactgatt cgggccgttc cgcatgttca ctttagaggc 3240
catgaagaat tccagtacct tgatctcatt gccgacatta ttaacaatgg aaggacaatg 3300
gatgaccgaa cgggcgttgg tgtcatctcc aaattcggct gcactatgcg ctactcgctg 3360
gatcaggcct ttccacttct caccacaaag cgtgtgttct ggaaaggggt cctcgaagag 3420
ttgctgtggt tcattcgcgg cgacacgaac gcaaaccatc tttctgagaa gggcgtgaag 3480
atctgggaca agaatgtgac acgcgagttc ctcgattcgc gcaatctccc ccaccgagag 3540
gtcggagaca tcggcccggg ctacggcttc cagtggagac acttcggcgc ggcatacaaa 3600
gacatgcaca cagactacac agggcagggc gtcgaccagc tgaagaatgt gatccagatg 3660
ctgagaacga atccaacaga tcgtcgcatg ctcatgactg cctggaatcc tgcagcgctg 3720
gacgaaatgg cgctgccgcc ttgtcacttg ttgtgccagt tctacgtgaa cgaccagaag 3780
gagctgtcgt gcatcatgta tcagcggtcg tgcgatgtcg gcctcggcgt ccccttcaac 3840
atcgcttcct attcgctttt gacgctcatg gttgcacacg tctgcaacct aaaacctaag 3900
gagttcattc acttcatggg gaacacgcat gtctacacga accatgtcga ggctttaaaa 3960
gagcagctgc ggagagaacc gagaccgttc cccattgtga acatcctcaa caaggaacgc 4020
atcaaggaaa tcgacgattt caccgccgag gattttgagg tcgtgggcta cgtcccgcac 4080
ggacgaatcc agatggagat ggctgtcttg gatcctgtcg ccaccatggt gagcaagggc 4140
gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 4200
cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 4260
aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc 4320
ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc 4380
aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 4440
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 4500
ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 4560
tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 4620
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 4680
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag 4740
tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 4800
accgccgccg ggatcactct cggcatggac gagctgtaca agaagcttag ccaaccggtc 4860
gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 4920
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 4980
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 5040
cccaccctcg tgaccacctt cggctacggc ctgcagtgct tcgcccgcta ccccgaccac 5100
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 5160
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 5220
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 5280
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 5340
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 5400
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 5460
aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 5520
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 5580
aagaagcttt aggcggccgc gtttgcagca gagtagttca tgacttgcga aacggcctct 5640
cgattaataa tacgctttgc cgcaacgtga agtaggcgtt attgtgtgct gcctgtcgct 5700
gagctcctgc atcgagcggc aaggggttca accgagcgca aattctgtgg aaatagctgg 5760
acaaaagcat ctgcgacggg tgggggcagg cgcagctagc tgttgctccc gacatcagtc 5820
atggaaatgc gtttcaggct aagcaagtag ctgcccgcct ggtgggaaga tgaggctagg 5880
aactgctatg tttgcccctc acttggcggt attgcagtgt agtacgtgca ttgtgaaccc 5940
agagaaatgt gctccgcgtg acgcagcgag gcacagggga acagcaagag gggccgttat 6000
tgctcagcgt gctggacccc cttgttcctt caaaccttac ttatgagggc ttacgcatcg 6060
cacctgatgg gtcggtgtag cgtgatcatt tcactgttca gtagtaggta tgggaggagt 6120
gtagttggca ggatgtagga gctttgcaag cggtggaacg gttgagatga gtagtgaaca 6180
gaggctttgc ccaatgtcgg atagtgagtg tttctcgagc acgaaacgtg caaggcaaca 6240
ggttactgta ggtttatgta gttggcaaga gtgggccttg cccagtattc ggtagtaagt 6300
gtcaagcacg acaccagatt gcgaagcaac aggtaattgt aggtttacgt atcgtacgtt 6360
tttcatggga gtgtgacgga aagttgtggg tagcagggtc cgggggagtc tgcggcaagc 6420
agttcaacag attgatggaa cagaaagatt aagaggcgga gtgtccgctg ttgctgtggg 6480
cagaaagagg gcggcgtaga gaggcattta gtggatgctt ttgaggagtt ctgggaggtc 6540
atcagtagtg gggaaggtct accgcaccgt ctggtcggcc tattagttaa tgcccacatg 6600
aggcgatctt ttggtggtgc gtgacggggt cagtcattgg atttggtatc tgccgctcca 6660
tttgtagcta ggagagcctc tgatgccgtg acatcttgct aggttcgcac tgtttccaca 6720
acgactactt tgtttggtca ttttcaccgt gaaagtatgt tcaccttgga gagagaaaat 6780
ttgagagagc tctgagttgt tggaacagcc caagtagctg actctcatag gatactacta 6840
aagccaaacg gcgcgttgta gcgctcatct gcagggattg atctttaaaa aaatgatctt 6900
ttaaggttca tgccatactt attgagttcg ctaggtgtca acttgtcgaa cacattcttg 6960
aggtgttgcg tatctgggcg ctcccagggg ttccatgtga agcaattgct tcacatggaa 7020
cccctgggag cgcccagata cgcaacacct caagaatgtg ttcgacaagt tgacacctag 7080
cgaactcaat aagtatggca tgaaccttaa aagatcattt ttttaaagat caatccctgc 7140
agatgagcgc tacaacgcgc cgtttggctt tagtagtatc ctatgagagt cagctacttg 7200
ggctgttcca acaactcaga gctctctcaa attttctctc tccaaggtga acatactttc 7260
acggtgaaaa tgaccaaaca aagtagtcgt tgtggaaaca gtgcgaacct agcaagatgt 7320
cacggcatca gaggctctcc tagctacaaa tggagcggca gataccaaat ccaatgactg 7380
accccgtcac gcaccaccaa aagatcgcct catgtgggca ttaactaata ggccgaccag 7440
acggtgcggt agaccttccc cactactgat gacctcccag aactcctcaa aagcatccac 7500
taaatgcctc tctacgccgc cctctttctg cccacagcaa cagcggacac tccgcctctt 7560
aatctttctg ttccatcaat ctgttgaact gcttgccgca gactcccccg gaccctgcta 7620
cccacaactt tccgtcacac tcccatgaaa aacgtacgat acgtaaacct acaattacct 7680
gttgcttcgc aatctggtgt cgtgcttgac acttactacc gaatactggg caaggcccac 7740
tcttgccaac tacataaacc tacagtaacc tgttgccttg cacgtttcgt gctcgagaaa 7800
cactcactat ccgacattgg gcaaagcctc tgttcactac tcatctcaac cgttccaccg 7860
cttgcaaagc tcctacatcc tgccaactac actcctccca tacctactac tgaacagtga 7920
aatgatcacg ctacaccgac ccatcaggtg cgatgcgtaa gccctcataa gtaaggtttg 7980
aaggaacaag ggggtccagc acgctgagca ataacggccc ctcttgctgt tcccctgtgc 8040
ctcgctgcgt cacgcggagc acatttctct gggttcacaa tgcacgtact acactgcaat 8100
accgccaagt gaggggcaaa catagcagtt cctagcctca tcttcccacc aggcgggcag 8160
ctacttgctt agcctgaaac gcatttccat gactgatgtc gggagcaaca gctagctgcg 8220
cctgccccca cccgtcgcag atgcttttgt ccagctattt ccacagaatt tgcgctcggt 8280
tgaacccctt gccgctcgat gcaggagctc agcgacaggc agcacacaat aacgcctact 8340
tcacgttgcg gcaaagcgta ttattaatcg agaggccgtt tcgcaagtca tgaactactc 8400
tgctgcaaac gcggccgcat ttgcggccgc ctactgaatg tcgccgctgt cggcccagta 8460
gtcgtcttcg tctgtgatgt cgataactgt atcggcttca ggtgtctcgg cagttgctgc 8520
tccggtatct tctccttcga actcgacctg ttctgcctcc tgtgcgccag cgccacctcc 8580
gccgctagtg aaggctgcaa caccaccacc tacagcagct atgagcaaca caccaccaac 8640
acctcctgcc actgcagctg ttgggaagcc gccgccttct tccttcttct cctcctcctc 8700
ttcgggttct gggcgttcgt gaccctcctc tggttttggc tggacggcag gagtttcggg 8760
ggcagcctca gactcctgct tgccctcttc ttcggtaccc tcaacaggct gctcaggtgt 8820
ctcctcaggc tgagtcacac cctcctcagg cttctcctca ggctcagtaa cctcctcctc 8880
aggtagctcc tcctcaggct tctcctcagg ctcagttaca ctctccccag gaagctcttc 8940
ttctgggggc tgcacttcgc cttctcctgt ttgctcgcct tcctcgccac ctgcttcacc 9000
agtttcgcct tcgcctgtct cggtctctcc ctctccttcg cctgtttccg taccgggggc 9060
tggaatgggg tgcaattcca tggtggcgac aggatcccgg gcccgcggga taccctggat 9120
gttgtcgcgc aacaccttgc ggtggcgctt ggcgcctccc tttcccaacc cctttcctcc 9180
ttttcctcgg ccagacattt tggttttcta tggaacagac acaacagaca gtctcgttgt 9240
agcctctaaa taacggatgt gagacatacg cgcgccgata ccatcacgaa caacaacacg 9300
cgcaccctgc acgtacagtg ggggaacatg cagtctctgc gcagctagcc tgctagcccg 9360
cgcccgggcg aacagccgac ccccagcctc gcttgttgtt tcagcaaata aagcgccatt 9420
gttttccaaa tattttattc tctatcctaa ttttttgcaa aattacgctt taataacagc 9480
ttaaggatgc aagtagaacc gacggacgtt attcaccgcc acctgtgcaa cgtacggaag 9540
tacacagggt ggtagaggag gtgctgcagc atccccgcgc ctcctccctc gctcgtagcc 9600
ttttggttaa aactaattct ttaaatgttt cttctgccaa ctgccatcaa gtttgtgctt 9660
ttaggactaa atacatccgt agattattgt tcagtccccg caggacttcg tttacgtcgc 9720
cttctgtata acaaccaccc cccacagttc aggtcatagc aacctactga gacgttgaga 9780
atatgcctgc atttgtgtgg agatatgcat tatttttttg agtgtctgaa gtgctgcaag 9840
cacgcatgac tatgcttcga aattaacggg ctgccacccg acagggtttg cgccgtttag 9900
gctgcttctg ctgctgctgg cacttctgtc tttgcatcgt ttagcaatat agtcgcgtga 9960
taaacaggct atgtacagca acgatgaaat ttattaatga caacagcact gcagatgcca 10020
tctgacggct aagtcactga atgttgcagt tgcagtggtg caagatacat gcgagagcgg 10080
aaagccgagc gcgagaagaa ggaactcgaa ctgttaaagt aggaggacct tagtgcgtcc 10140
cacttactgc ggggcacagt acaaacgccg gggactacag ctacgagctc gttcgtttgt 10200
aaacccgggg tgacggaaga attggcgaaa agagcagtgg aggtgctaat ccactgctgc 10260
cccaaattcc cttgaggccc taaccaaaaa agtgcgagca aatgcccgtc tgtgtgagta 10320
acagtgattc gatggtttca cttgccaatg gctgttaaag gcagtccagc tcctgcttgc 10380
tcgaactgcc gccttccaaa agctctttac ggaggtcctt aacaccagtg gtaatggcaa 10440
actctaaatg tattggtcac actagcggaa gcttctggca gcaaaaccag tacgcctatt 10500
gagagtcaca cacatcccct tagaggggag ctttttgcat gctttgcccc gcaggttgca 10560
cagcattttg catgatgcat gcttttccca gtagagagtt cagctgcagc agcatcagct 10620
gcatgggctt tatatgcttc tgtaactgct gctgttgcgt ggctttgaaa acaacagggc 10680
tgggtattca aatactcact agaacgctgc tgttacgata tcatcaactg cgcgagcaac 10740
tatttggaaa ggaagagggc gggacattca agcacatcct agaatgctgt tgtagagata 10800
ttatcacctg ggggagcagc tatttgtgcc gctgtttttt tctgcaacta accgcagcga 10860
atgggcaaag cagccacaat gagtcgagca tcagccgttt cgcggcggat ggctcaccac 10920
gacagcaata gcgggttcta taagaattta aaaaacagaa aaagcaacag agtctccaac 10980
aatggaccaa tagatgtcag cagagcaaac agaaaccgcg aatgattcag ttttacagaa 11040
gctatgtatc tcgagtgtaa caagttcgtt cttgtgcctg tcactgacgg tgacaaaggt 11100
aacattgcta acctctactg ttgctacctt tgctggttca taaacttgtt tattgcagct 11160
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 11220
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag 11280
cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 11340
ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 11400
tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 11460
gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 11520
tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 11580
agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 11640
gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 11700
gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac 11760
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 11820
ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg 11880
tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg 11940
caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca 12000
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact 12060
ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 12120
atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 12180
tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg 12240
atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 12300
gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 12360
ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 12420
gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 12480
cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 12540
gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc 12600
tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 12660
gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 12720
tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag 12780
catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat 12840
ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg 12900
ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc 12960
tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta 13020
tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg 13080
acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 13140
ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 13200
gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa 13260
ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt 13320
gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc 13380
cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt 13440
gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac 13500
tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 13560
atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 13620
tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 13680
tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 13740
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 13800
cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 13860
ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 13920
gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 13980
tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 14040
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 14100
ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 14160
tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 14220
ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 14280
tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 14340
attaccgcca tgcat 14355
Claims (10)
1、球虫在活疫苗制备中的应用。
2、如权利要求1所述的应用，其中所述的球虫为禽类艾美耳科球虫或哺乳动物艾美耳科球虫。
3、如权利要求2所述的应用，其特征在于，所述的球虫为艾美耳科球虫。
4、一种球虫载体活疫苗，其以球虫作为免疫活载体表达目的蛋白。
5、如权利要求4所述的球虫载体活疫苗，其为能表达外源蛋白的转基因球虫或为含有表达载体可以表达目的蛋白的稳定转染球虫。
6、如权利要求5所述的球虫载体活疫苗，其中所述的转基因球虫为被打靶或随机整合到其基因组中能时空调控外源蛋白表达的转基因球虫。
7、如权利要求5所述的球虫载体活疫苗，其中所述的表达载体为pHDEA。
8、如权利要求4～7任一项所述的球虫载体活疫苗，其中所述的目的蛋白为病原体抗原蛋白
9、如权利要求8所述的球虫载体活疫苗，其特征在于所述的病原体蛋白为巨型艾美耳球虫TFP250蛋白、禽流感病毒HA、NA、NP、M1、M2、NS1、NS2、PA、PB1和PB2蛋白、鸡新城疫病毒F蛋白。
10、权利要求4～7任一项所述的球中载体活疫苗，其中所述的球虫为艾美耳科球虫。
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100649248A CN101024076B (zh) | 2007-03-29 | 2007-03-29 | 球虫的新用途 |
| US12/593,851 US8142771B2 (en) | 2007-03-29 | 2008-03-28 | Use of coccidian |
| PCT/CN2008/000621 WO2008119254A1 (en) | 2007-03-29 | 2008-03-28 | New use of coccidian |
| EP08733851.3A EP2140877B1 (en) | 2007-03-29 | 2008-03-28 | New use of coccidia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100649248A CN101024076B (zh) | 2007-03-29 | 2007-03-29 | 球虫的新用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101024076A true CN101024076A (zh) | 2007-08-29 |
| CN101024076B CN101024076B (zh) | 2010-08-18 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2007100649248A Active CN101024076B (zh) | 2007-03-29 | 2007-03-29 | 球虫的新用途 |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US8142771B2 (zh) |
| EP (1) | EP2140877B1 (zh) |
| CN (1) | CN101024076B (zh) |
| WO (1) | WO2008119254A1 (zh) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008119254A1 (en) * | 2007-03-29 | 2008-10-09 | China Agricultural University | New use of coccidian |
| CN102657865A (zh) * | 2012-04-28 | 2012-09-12 | 中国农业大学 | 利用转基因球虫向哺乳动物肠道靶向运送蛋白或抗原的方法 |
| CN103233017A (zh) * | 2013-05-06 | 2013-08-07 | 扬州大学 | 毒害艾美耳球虫的配子体抗原gam22基因及其应用 |
| CN104593406A (zh) * | 2015-01-08 | 2015-05-06 | 山西医科大学 | pIRES/TgDHFR-TS真核表达重组质粒及其构建和应用 |
| CN104673830A (zh) * | 2014-01-28 | 2015-06-03 | 中国农业大学 | 一种提高禽畜钙磷利用率的方法 |
| CN104830882A (zh) * | 2014-02-10 | 2015-08-12 | 中国农业大学 | 表达鸡IgY分子Fc片段和禽流感病毒抗原的融合基因及其应用 |
| CN104940924A (zh) * | 2015-05-29 | 2015-09-30 | 中国农业大学 | 一种球虫佐剂及其应用 |
| CN106267175A (zh) * | 2016-09-19 | 2017-01-04 | 中国农业大学 | 一种鸡球虫病疫苗及其应用 |
| CN106421767A (zh) * | 2016-09-19 | 2017-02-22 | 中国农业大学 | 具备交叉免疫保护力的球虫病疫苗组分及疫苗 |
| CN106492208A (zh) * | 2016-09-19 | 2017-03-15 | 中国农业大学 | 一种鸡球虫病疫苗免疫方法 |
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| CA3156538C (en) * | 2007-11-01 | 2023-12-12 | The Board Of Trustees Of The University Of Arkansas | Compositions and methods of enhancing immune responses to eimeria |
| AR089868A1 (es) | 2012-02-03 | 2014-09-24 | Animal Health Inst | Vacuna vectorial de eimeria para campylobacter jejuni |
| NZ711019A (en) | 2013-02-14 | 2019-07-26 | Univ Arkansas | Compositions and methods of enhancing immune responses to eimeria or limiting eimeria infection |
| CA2948668C (en) | 2014-05-09 | 2023-06-13 | View, Inc. | Control method for tintable windows |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976553A (en) * | 1993-11-04 | 1999-11-02 | The Board Of Trustees Of The Leland Stanford Junior University | Transfection and genetic manipulations in obligate intracellular parasites |
| US20030175287A1 (en) * | 2001-01-03 | 2003-09-18 | Yale University | Innate immune system-directed vaccines |
| RU2324498C2 (ru) | 2002-12-09 | 2008-05-20 | Юниверсити Оф Джорджия Рисерч Фаундейшн, Инк. | Кокцидиальная вакцина и методы ее приготовления и использования |
| US7722860B2 (en) * | 2004-12-03 | 2010-05-25 | Seattle Biomedical Research Institute | Live genetically engineered protozoan vaccine |
| CN100998866A (zh) | 2006-01-09 | 2007-07-18 | 佛山市正典生物技术有限公司 | 一种由药物敏感虫株组成的鸡球虫病四价活疫苗的制备方法 |
| CN101024076B (zh) * | 2007-03-29 | 2010-08-18 | 中国农业大学 | 球虫的新用途 |
-
2007
- 2007-03-29 CN CN2007100649248A patent/CN101024076B/zh active Active
-
2008
- 2008-03-28 EP EP08733851.3A patent/EP2140877B1/en active Active
- 2008-03-28 US US12/593,851 patent/US8142771B2/en active Active
- 2008-03-28 WO PCT/CN2008/000621 patent/WO2008119254A1/zh active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008119254A1 (en) * | 2007-03-29 | 2008-10-09 | China Agricultural University | New use of coccidian |
| CN102657865A (zh) * | 2012-04-28 | 2012-09-12 | 中国农业大学 | 利用转基因球虫向哺乳动物肠道靶向运送蛋白或抗原的方法 |
| CN103233017A (zh) * | 2013-05-06 | 2013-08-07 | 扬州大学 | 毒害艾美耳球虫的配子体抗原gam22基因及其应用 |
| CN103233017B (zh) * | 2013-05-06 | 2014-11-05 | 扬州大学 | 毒害艾美耳球虫的配子体抗原gam22基因及其应用 |
| CN104673830A (zh) * | 2014-01-28 | 2015-06-03 | 中国农业大学 | 一种提高禽畜钙磷利用率的方法 |
| CN104830882A (zh) * | 2014-02-10 | 2015-08-12 | 中国农业大学 | 表达鸡IgY分子Fc片段和禽流感病毒抗原的融合基因及其应用 |
| CN104830882B (zh) * | 2014-02-10 | 2018-02-06 | 中国农业大学 | 表达鸡IgY分子Fc片段和禽流感病毒抗原的融合基因及其应用 |
| CN104593406A (zh) * | 2015-01-08 | 2015-05-06 | 山西医科大学 | pIRES/TgDHFR-TS真核表达重组质粒及其构建和应用 |
| CN104940924A (zh) * | 2015-05-29 | 2015-09-30 | 中国农业大学 | 一种球虫佐剂及其应用 |
| CN104940924B (zh) * | 2015-05-29 | 2018-01-16 | 中国农业大学 | 一种球虫佐剂及其应用 |
| CN106267175A (zh) * | 2016-09-19 | 2017-01-04 | 中国农业大学 | 一种鸡球虫病疫苗及其应用 |
| CN106421767A (zh) * | 2016-09-19 | 2017-02-22 | 中国农业大学 | 具备交叉免疫保护力的球虫病疫苗组分及疫苗 |
| CN106492208A (zh) * | 2016-09-19 | 2017-03-15 | 中国农业大学 | 一种鸡球虫病疫苗免疫方法 |
| CN106421767B (zh) * | 2016-09-19 | 2019-07-26 | 中国农业大学 | 具备交叉免疫保护力的球虫病疫苗组分及疫苗 |
| CN106492208B (zh) * | 2016-09-19 | 2019-11-26 | 中国农业大学 | 一种鸡球虫病疫苗免疫方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101024076B (zh) | 2010-08-18 |
| EP2140877B1 (en) | 2015-01-21 |
| WO2008119254A1 (en) | 2008-10-09 |
| US8142771B2 (en) | 2012-03-27 |
| EP2140877A4 (en) | 2010-04-21 |
| US20100183668A1 (en) | 2010-07-22 |
| EP2140877A1 (en) | 2010-01-06 |
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