具体实施方式
以下实施例用于进一步说明本发明,但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下,对本发明所作的修饰或者替换,均属于本发明的范畴。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
1、豚草花粉DNA提取
采用天根快速DNA提取扩增试剂盒(天根生化KG203)。
提取方法:称取5mg豚草花粉样品置于1.5mL离心管中,加入Buffer 1100μl后用一次性研磨杵将样品彻底研碎后加入Buffer 2 100μl后震荡混匀,离心机12000r/min离心5min后取上清液于新的离心管中作为DNA模版备用。
2、引物设计及合成
引物序列如下:
Amb ITS2-F(SEQ ID NO.1):5'-GGAAGTAAAAGTCGTAACAAGG-3'
Amb ITS2-R(SEQ ID NO.2):5'-TCCTC CGCTTATTGATAT GC-3'
引物序列由上海生工生物工程有限公司合成。合成后分装-20℃保存。
3、PCR反应体系
PCR扩增试剂盒(天根生化)。对提取的DNA采用如下50μl体系的配制PCR反应体系见表1:
表1.豚草花粉ITS2序列PCR反应体系
4、PCR反应条件见表2.
表2.豚草花粉ITS2序列PCR反应条件
对PCR扩增后的反应液进行2%琼脂糖电泳,150V、100mA 20min电泳观察,电泳结果如图1所示。其中L1、L2、L3为3批豚草花粉PCR产物,Lmix是上述3批豚草花粉混合原料PCR产物,DNA Marker如图所示。结果显示,PCR产物在720bp左右。
5、测序
利用PCR产物纯化回收试剂盒(生工SK1141),回收PCR产物进行测序,测序结果编号Amb ITS2,如SEQ ID NO.3所示。
6、豚草的显微镜检
豚草花粉显微镜下形态学鉴别:三裂叶豚草花粉球形。直径19(18~20)微米。极面观三裂圆形,赤道面观圆形;具3孔沟。
实施例2豚草花粉脱脂及浸提关健工艺步重要参数的确定
1、豚草花粉脱脂工艺步参数确定
(1)将豚草花粉(g)与丙酮(ml)以1:2的重量体积比(w/v)进行脱脂处理。对不同批次(不同采集时间)的豚草花粉不同脱脂时间样品进行脂肪含量进行检测,以确定最优的脱脂时间。
采用海能SOX500脂肪测定仪测定豚草花粉中脂肪含量,通过索式萃取及干燥称重的方法,对比萃前后花粉重量的变化,得到相对应的脂肪含量,并根据脂肪含量结果对之后脱脂后花粉进行质量控制.
结果见图2,可见丙酮脱脂时间为30min后,脂肪含量几乎不随脱脂时间延长而下降,因此脱脂时间确定为30min。
(2)脱脂参数的验证:对不同批次(不同采集时间)的豚草花粉进行30min脱脂后检测脂肪含量,结果如表3所示。
表3.不同批次豚草花粉脱脂30min后脂肪含量降低百分比
从结果得知,豚草花粉中脂肪含量在1.5%~2%,故在之后脱脂过程中,脱脂后的豚草花粉在脂肪含量上应该下降1.5~2%。
2、豚草花粉蛋白浸提工艺步重要参数确定
(1)将豚草花粉(g)与丙酮(ml)以1:2的重量体积比(w/v)进行搅拌或振荡脱脂30分钟处理,静置分层,观察上层液体澄清情况,重复脱脂至上层液体澄清后,将脱脂后的花粉均匀摊开,室温下干燥48小时以上。
(2)将脱脂干燥后的豚草花粉按重量体积比1:10加入10L,pH 7.9~8.2磷酸盐—盐水缓冲液,2~8℃,在搅拌速度为250rpm条件下,在搅拌时间分别为3h、6h、9h、12h、18h、24h、48h、72h等提取点时,将提取后的提取液,将浸提液全部加入到离心桶内,调节平衡,离心力8000~12000g,离心温度4℃,时间设定为15~20分钟,进行离心,收取上清。
(3)将离心分离后的上清液先用4000目滤布过滤后,通过纸板过滤,0.45μm和0.22μm滤膜依次过滤;得到豚草花粉粗提取液。
取样以Bradford法测定蛋白含量,结果见“图3、豚草花粉浸液总蛋白含量与浸提时间的关系”。
如图3结果表明,在蛋白提取过程中,随搅拌提取时间延长,蛋白提取含量增大,提取时间24h含量达到最高1406±13μg/ml,可见蛋白类物质提取时间过长会影响其蛋白含量,故提取时间优选为22~26h左右。
实施例3豚草花粉原料工艺
1、花粉采集
自然脱落法,收集豚草花粉。常温或真空或流化床干燥,直至花粉不再粘附。将干燥后的花粉过150~250目分样筛,本实施例中优选为180目分样筛。
2、干燥
将花粉摊放于通风干燥处自然干燥,或真空干燥、或流化床干燥6~48h,直至花粉不再粘附。
3、脱脂
将上述所得花粉(g)与丙酮(ml)以1:5~1:1的重量体积比(w/v)进行脱脂处理。本实施例中优选为1:2。搅拌或振荡脱脂30分钟后,静置分层,观察上层液体澄清情况。倒出上层液,加入新的丙酮,重复脱脂至上层液体澄清。
4、再干燥
将脱脂后的花粉均匀摊开,室温干燥、或真空干燥、或流化床干燥6-48h。
5、杂质控制
(1)显微镜下用颗粒计数法测定,其中的杂质颗粒含量应满足如下标准:孢子含量≤1%,无关花粉含量≤2%,其它杂质含量≤10%。
(2)重金属及有害元素
总重金属≤50mg/kg;砷≤5mg/kg。
(3)丙酮残留
丙酮残留量≤0.5%(体积分数)。
实施例4豚草花粉变应原原液(冻干品)制备工艺
1、按实施例3豚草花粉原料工艺制备豚草花粉原料。
2、浸提
将脱脂干燥后的豚草花粉按重量体积比1:50~1:10加入10L,本实施例中优选为1:20,以pH 7.9~8.2磷酸盐—盐水缓冲液,2~8℃搅拌22~26h进行浸提。其中以配制1000ml磷酸盐—盐水缓冲液(pH 8.2)为例的配方如表4。磷酸盐—盐水缓冲液充分溶解后除菌过滤,2~8℃放置,保存期不超过30天。
表4磷酸盐—盐水缓冲液(pH 8.2)配方
3、固液分离
取提取后的浸提液,将浸提液全部加入到离心桶内,调节平衡,离心力8000~12000g,离心温度2~8℃,时间设定为15~20分钟,进行离心,收取上清;
4、澄清
将离心分离后的上清液先用4000目滤布过滤后,将滤液依次通过纸板过滤、0.45μm和0.22μm滤膜依次过滤;
5、超滤、透析、浓缩
过滤后的浸提液,用3KD或1KD超滤膜切向流超滤,优选地,本实施例中用3KD超滤膜超滤。透析液选用25-125mM的NH4HCO3,本实施例中优选为50mM的NH4HCO3。根据蛋白含量质量标准,超滤浓缩至适当体积,检测蛋白含量至质量标准的总蛋白含量区间。取样超滤浓缩液、超滤透过液,检测其总蛋白含量。当超滤透过液中总蛋白含量≤0.02mg/ml,则直接排弃透过液;超滤透过液总蛋白含量>0.02mg/ml,则对超滤膜进行完整性测试,超滤膜未破损,则排弃透过液;若超滤膜破损,则更换超滤膜之后对透过液重复超滤。
6、无菌过滤
用0.22μm膜除菌过滤。
7、冻干
按照冻干工艺条件,-50~-35℃冻结,2~8mbar真空压力下,-25℃干燥,控制水分含量≤3%;获得豚草花粉变应原冻干品。
实施例5豚草花粉变应原浸液成品制备工艺
1、复溶
将按实施例4制备的豚草花粉变应原冻干品用磷酸盐—盐水缓冲液(pH6.5~7.5,配方见表5)复溶,至总蛋白含量位于质量标准2倍范围区间。2~8℃放置。
表5.磷酸盐—盐水缓冲液(pH值6.5~7.5)配制
2、半成品配制
原液复溶后,在净化车间内A级洁净条件下,将复溶液与等体积预先湿热灭菌(121℃、30分钟)并冷却的甘油混匀,调节溶液pH值至6.0~8.0。
3、半成品除菌过滤、无菌分装为成品
在净化车间内A级洁净条件下,将半成品通过0.22μm滤膜除菌过滤,无菌分装为成品,得到pH6.0~8.0浅黄色至棕色液体即为豚草花粉标准化变应原浸液成品。
实施例6豚草花粉变应原浸液成品质量控制
1、豚草花粉标准化变应原浸总蛋白成分SDS-PAGE法鉴别
采用还原型SDS-PAGE法,分离胶浓度为4~12%,考马斯亮蓝染色。结果如图3所示。M为Genstar M223-01Marker,R为冻干内部参考品(即冻干的豚草花粉变应原原液),L1,L2和L3为不同批次豚草花粉标准化变应原浸液,其蛋白分布主要在10kDa、14kDa、18kDa、37kDa、38-42kDa、43.8kDa、48kDa、52kDa、68kDa左右,其中鉴定的致敏蛋白分子量、过敏血清阳性率、对应全蛋白质谱鉴定肽段概览见表6。
2、豚草花粉标准化变应原浸总变应原成分Western Blotting法鉴别
采用还原型SDS-PAG电泳,分离胶浓度为4~12%,0.2μm PVDF ImmobilonR-PSQ转印膜,一抗血清反应稀释度1:3,室温杂交2h,随后置于4℃冰箱杂交过夜,1×PBST洗3次,10min/次。。二抗Ms mAb to Hu IgE(ab99806)1:1000室温杂交2h,1×PBST洗3次,10min/次。将ECL显影液A液和B液1:1混匀后曝光显色。
结果见图4所示。其中,M为Genstar Marker,R为内部参考品,N为浸液与健康受试者血清反应条带,P1-24分别为浸液与28例临床确诊的豚草过敏阳性患者(sIgE≥3级,皮试≥+++,典型的夏秋季花粉症病史)血清反应条带结果,其中致敏Amb t 1.04’、Amb t1.05’、Amb t 6、Amb t 8、Amb t 10、Amb t 11、Amb t 12、Amb t X1、Amb t X2的分子量及过敏血清阳性率汇总如表6,由此确定血清阳性率最高的Amb t 1.04’(38-42kDa)、Amb t1.05’(43.8kDa)为豚草花粉的主要致敏蛋白(血清阳性率最高,为96.4%)。
3、致敏蛋白序列测定
本发明产品对相应致敏蛋白(见表6)进行了全蛋白序列测定,并对三裂叶豚草花粉原料中每一致敏蛋白对应mRNA进行了核苷酸全长测序,致敏蛋白代码、序列标识及氨基酸全长序列、对应mRNA全长序列结果详见表6。
其中本发明所含三裂叶豚草致敏蛋白的全序列依次为:
Amb t 1.04’致敏蛋白全序列为SEQ ID NO.4:
CCYILYFTLALVILLQPVRSAEDLQQILPSVNETRSLTTCEANNIIDKCWRCKPDWAENRKALADCAQGFAKGTIGGKMGDIYTVTSDLDDDVANPKEGTLRFGAAQNRPLWIIFARDMVIRLDKEMEVNNDKTIDGRGARVEIINAGFRIKEVKNVIIHNIYFHDINAQPGALVKRNEGPPRLRKGSDGDAISLDGCSQVWIDHCSLSKAFDGLVDAKLGTTRFTVSNSLFTQHQYVLLFWDFEETGMLGTVAFNKFTDHVDQRMPNCRHGFFQVVNNNYDRWGSYCIGGSAAPTILSQGNRFFAPDDPIKKNAVGRHGEAAQESMKWNWRSYNDLLENGAIFVASGVDPVLTPEQSEGMILAEPGEAVLRLTSSAGVLSCQPGAPC
Amb t 1.05’致敏蛋白全序列为SEQ ID NO.5:
MGIKHCCYILYFTLALVTLVQAGRLGEEVDILPSPNDTRRSLQGCEAHNIIDKCWRCKPDWAENRKALGDCAKGFGKGTYGGKGGDIYMVTSDQDDDVVNPREGTLRFGATQDRPLWIIFERDMIIYLQQEMVVTSDTTIDGRGAKVELVYGGITLMNVKNVIIHNLNIHDVRVLPGGLIKSSGGPAIPRYKSDGDAVHVTGSSDIWIDHCTLGNSFDGLVDVNWGSSSVTISNCKFERQDKAILLGASDTHFQDVKMHVTLAYNIFANTVHERMPRCRFGFTQIVNNFYDRWDKYAIGGSSNPTILSQGNKFVAPDNIYRKNVCLRTGAQEPEWMTWNWRTQNDLLENGAVFVASGSDPVLTAEQNAGMIQAEPGETVPQLTMNAGVLTCSPGAPC
Amb t 8致敏蛋白全序列为SEQ ID NO.6:
MSWQTYVDEHLMCDIEGTGQHLASAAIFGTDGNVWAKSTSFPELKPDEINAIIKEFSEPGTLAPIGLFLAGAKYMVIQGESGAVIRGKKGAGGICIKKTGQAMVFGIYEEPVNPGQCNMVVERLGDYLVDQGM
Amb t 10致敏蛋白全序列为SEQ ID NO.7:
MAPENNKLSIFPTDKEEITKIFNRFDTNGDGQISKEELFDILKSLGSDTSPDEVKRVMAEIDADGDGFISLDEFVLFCKGMEAEGDEMNDLKEAFKLYDQNNNGVISANELHQILGRLGENYTVERCADMIKSVDSDGDGFVDFEEFRKMMSRKGGDGAH
Amb t 11致敏蛋白全序列为SEQ ID NO.8:
MEINKFVSFSFSLVLILGLAESFHYHERELESDEGFLGMYDRWREQHNIEMRSPERFNVFKYNVKRIHESNKMDKPYKLKVNEFADMTNLEFVNTYANSKISHFQALRGGAPGSLDTDPNKDFIYANATKIPDKLDWREKNAVTDVKGQGGCGSCWAFAAVVALEGINAIRTGKLVKFSEQQLVDCDMTNAGCDGGLMEPAFTYVIKHGGIAPEANYPYVGKRETCDKAKIKDVLKIDGRQNVPGLDEELLRKAVAHQPVATGIQLSGHGLQFYSEGVYTGDCGTEPNHGVGIVGYGENDKGIKFWTVKNSWGPTWGEKGYIHLQRGARKEGLCGIAMHSSFPIMNDPNPPKDDPIKAPKDDPDLPKDPKFKTTQRLQGIRTKLLEL
Amb t 12致敏蛋白全序列为SEQ ID NO.9:
MATIKAVKARQIFDSRGNPTVEVDVTLSDGTLARAAVPSGASTGIYEALELRDGGSDYLGKGVLKAVDNVNSIIGPALIGKNPTEQTKIDNFMVQELDGTVNEWGWCKQKLGANAILAVSLAVCKAGASVKKIPLYKHIANLAGNKTLVLPVPAFNVINGGSHAGNKLAMQEFMILPVGASSFKEAMKMGVEVYHTLKSVIKKKYGQDATNVGDEGGFAPNIQENKEGLELLKTAISKAGYSGKVVIGMDVAASEFYGEKDKTYDLNFKEENNDGKEKISGEQLKDLYKSFVSEYPIVSIEDPFDQDDWEHYAKMTAECGEQVQIVGDDLLVTNPTRVKKAIDEKTCNALLLKVNQIGSVTESIEAVRMSKHAGWGVMASHRSGETEDTFIADLSVGLATGQIKTGAPCRSERLAKYNQLLRIEEELGSEAVYAGANFRKPVEPY
通过对三裂叶豚草花粉原料中上述每一致敏蛋白对应mRNA进行了核苷酸全长测序后,其mRNA对应序列如下
Amb t 1.04’致敏蛋白基因全序列为SEQ ID NO.10:
tgttgttacatcttgtattttaccttagcccttgtcattttgctgcaacctgttcgttctgccgaagatctccagcaaatcttaccttcagttaacgaaacaaggagcctgacaacatgtgaagcaaacaacattatagacaagtgctggaggtgcaaacctgattgggcagaaaaccgaaaagcgttagccgattgtgcccaaggttttgcaaagggaaccatcggcggaaaaatgggtgatatctacacggtgaccagtgatctagatgatgatgttgctaatccaaaagaaggcacactccggtttggtgccgcccaaaacaggcccttgtggattatttttgcaagagatatggtgattcgtttggataaagagatggaggtaaacaacgacaagaccatcgatggccgaggggcgagagttgaaatcattaacgctggtttccgcatcaaagaagtcaagaatgtaatcattcataacatatattttcatgatattaatgcgcagccaggagccctggttaagagaaacgaaggtccaccacgtctaagaaagggtagtgatggtgatgctataagtcttgatggttgttcacaagtatggatcgaccattgctcgctcagtaaggcttttgatgggctagtcgatgccaaactcggcaccacacgcttcaccgtttccaacagcttatttacccaacaccaatatgtattattgttttgggattttgaagaaacaggcatgctaggaacggtcgcattcaacaagttcactgatcacgttgaccaaagaatgcctaattgtcggcatgggtttttccaagtcgttaacaacaactacgatagatggggatcgtattgcatcggtggtagcgcggccccaaccatactcagccaaggaaacagattctttgcccccgatgatcctatcaagaaaaatgctgtagggaggcatggtgaagccgcacaagagtcgatgaagtggaactggagatcgtataatgatctgcttgaaaatggtgctatttttgttgcatccggcgttgatccagtgttaacccctgaacaaagcgaagggatgattctagccgaaccaggtgaagccgttctaagactcactagtagtgccggtgtactctcatgccaacctggagcaccttgctaa
Amb t 1.05’致敏蛋白基因全序列为SEQ ID NO.11:
atggggatcaaacattgttgttacatcttgtattttaccttagcacttgtcactttggtgcaagctggacgtcttggcgaagaggtcgacatcttaccttcacctaacgatacaaggaggagcctgcaaggatgtgaagcacacaacattatagacaagtgttggaggtgcaaacccgattgggcggagaaccgaaaagcgttaggcgattgtgccaaaggttttggaaagggaacttacggcggaaaagggggtgatatctacatggtcacaagtgatcaggatgatgatgttgtgaatccaagagaaggcacactccggttcggtgccacccaagacaggcccttgtggatcatttttgaaagagatatgattatttatttgcaacaagagatggtagtaaccagcgacacgaccatcgatggtcgaggggcgaaagttgagcttgtttatggaggtatcaccctcatgaatgtcaagaatgtaatcattcacaacctaaatatccatgatgttagagtgcttccaggaggcctgattaagtccagcggtggtccagcaataccaagatataagagtgatggtgatgctgtccatgttactggtagttcagacatatggatcgaccattgcacgctcggcaattcatttgatgggctcgtcgatgtcaactggggtagctcatcagtaaccatttccaactgcaaattcgaacgccaagacaaagccattttgctcggggctagtgacacccattttcaagatgtgaaaatgcatgtaacgcttgcatacaacatcttcgccaataccgttcatgaaagaatgcctagatgccgatttgggtttacccaaattgtaaacaacttctacgacagatgggataagtacgccattggtggtagctcgaaccctactattctcagccaagggaacaaattcgtggcccccgataacatttacaggaaaaacgtctgtctgaggactggtgcacaggagccagaatggatgacttggaactggagaacgcaaaacgacctgcttgaaaatggtgctgtctttgtggcatccgggtctgatccagtgctaaccgctgaacaaaatgcaggcatgattcaagctgaacctggagaaacggttccgcaactcaccatgaatgctggtgtactcacatgctcgcctggagcaccttgctaa
Amb t 8致敏蛋白基因全序列为SEQ ID NO.12:
atgtcgtggcagacttacgtggatgaacatttgatgtgtgacatcgaaggcactggtcagcatctcgcctctgccgctatcttcggtaccgatggcaacgtctgggccaagagcacttctttccccgagttgaaacctgatgagattaatgctatcatcaaagaattcagtgaacctggtactcttgcccctattggtttattcctagccggtgcaaagtacatggtgatccaaggcgagtctggagctgttattcgtggaaagaagggagctggagggatctgcatcaagaaaactggtcaagccatggtgtttggcatctatgaggagccggtgaatcctggtcagtgcaacatggttgtggaaaggttgggtgattatcttgtggatcaaggcatgtaa
Amb t 10致敏蛋白基因全序列为SEQ ID NO.13:
atggcgccagaaaataacaaactttcaatattcccaacagacaaagaagaaataacaaaaatcttcaaccgtttcgacacaaacggcgacggccaaatctccaaagaagagctattcgacatcctgaaatcgctcggttctgatacatctcctgacgaggtgaagcgagtaatggctgaaatcgatgcagatggcgacggtttcattagccttgatgagtttgttctattctgcaaaggaatggaagccgaaggcgatgagatgaatgatctcaaggaagcgtttaagttgtatgatcagaacaacaatggtgtgatatctgcaaatgaattgcatcagatattgggtcggttgggagagaattacacggttgaaaggtgcgcggatatgattaagtctgttgattcggatggcgatggttttgttgattttgaagagtttaggaagatgatgtcaaggaagggaggtgatggtgcacattaa
Amb t 11致敏蛋白基因全序列为SEQ ID NO.14:
atggaaatcaacaagtttgtttctttttcattttctttggttttgattttaggacttgcggagagcttccattaccatgagagagagctcgaatcggacgaggggtttttggggatgtacgataggtggagggagcaacacaatatcgaaatgagaagcccagaacggttcaatgtattcaaatacaatgttaaacgcattcacgaatcgaataagatggacaagccgtataagttgaaggtgaacgagtttgctgacatgactaaccttgagtttgttaacacgtatgctaactcgaagattagccattttcaagctctccgaggaggagcacctggatcgcttgataccgaccctaataaagatttcatatatgcaaatgcgactaaaatcccagataagcttgattggagagagaaaaatgccgtcactgatgtcaagggtcaaggcggatgtggaagttgttgggcatttgccgctgtggttgcattggaaggaataaacgcgatcagaaccggaaaactagtaaaattttccgaacaacaactcgtcgactgtgacatgacgaacgcaggatgcgacggagggctaatggaacctgcattcacatacgtcataaagcatggaggtatagcaccagaagccaactacccttacgtaggcaaaagagaaacctgcgacaaagcaaagattaaagatgtgttgaagatcgatggtagacaaaatgtgcctggacttgatgaagaattactaaggaaggcagtagcacaccagcctgtagctacaggcatacaacttagcggccatggtttgcagttctattccgagggtgtatataccggagattgtggtacagagccgaatcatggagttggaattgtgggatacggtgagaatgataaggggattaaattttggaccgtgaagaactcatggggaccaacgtggggagagaagggatacatacatctacaacgcggagctaggaaagagggactctgcggaatagcaatgcattcttcttttcctattatgaacgacccaaatccacctaaagacgaccccattaaagcacctaaagacgaccctgatttacctaaggaccctaaatttaaaacgactcagaggttgcaggggataaggactaaattgttggagttgtga
Amb t 12致敏蛋白基因全序列为SEQ ID NO.15:
atggcgacgatcaaagccgttaaagctcgtcagatcttcgatagtcgtggtaatcctacggtcgaagttgatgttactctgtctgatggaactttggctagagccgctgtacctagcggtgcttctacaggtatatatgaagcgttggagttgcgagatggaggttcggattatctcggtaaaggtgtcttgaaggctgtggacaatgttaactcgattattggtcctgccttgattggcaagaatcctacggaacagaccaaaattgacaacttcatggttcaagagcttgatggaactgtaaatgaatggggctggtgcaaacagaagcttggtgccaatgccatactggcagtgtcacttgctgtgtgtaaagctggagcatctgttaagaaaattcctctatataagcacattgcaaacctagctggaaacaagacactggtgttgccagtaccagcatttaatgtgatcaatggtgggtcgcatgcaggcaacaaacttgcaatgcaagagtttatgattctccctgttggagcttcttccttcaaggaagccatgaagatgggtgttgaagtgtatcacaccttgaagtctgtaattaaaaagaaatatggtcaagatgctaccaatgttggtgatgaaggtggatttgctcctaacatccaggaaaacaaagaaggccttgaacttttgaagactgcaatatcaaaagctggatacagcggaaaggttgttattggaatggatgttgctgcatctgaattctatggcgagaaggacaagacctatgatctgaacttcaaggaagagaacaacgatggcaaagaaaagatttcaggagaacaactaaaagatctttacaagtcgtttgtgagtgagtaccccattgtgtccattgaggacccatttgaccaagatgactgggagcactatgccaagatgaccgctgaatgtggcgaacaagttcaaattgtaggagacgatcttttggtcaccaaccccacgagagtcaagaaggcaattgatgagaagacttgcaatgctcttcttctaaaggtcaaccaaattgggtctgtgaccgagagtatcgaagctgtgaggatgtccaaacatgctggttggggtgtgatggccagtcaccgcagtggagaaacagaggacaccttcattgctgatctttctgtgggtttggcaacgggtcaaatcaaaactggagctccatgcaggtcagagcgtcttgcaaagtacaaccagctgttgagaatcgaagaagagcttggatcagaagcggtttatgctggagccaacttccgcaagccagtggaaccctactag
4、质谱检测
对豚草变应原浸液进行全蛋白质谱分析以及对豚草变应原经SDS-PAGE分离后对应的胶条分别取样进行质谱鉴定,其主要包含且不限于以下肽段:
Amb t 1.04’致敏蛋白质谱鉴定肽段:
SEQ ID NO.16:MGDIYTVTSDLDDDVANPK
SEQ ID NO.17:KALADCAQGFAK
SEQ ID NO.18:LDKEMEVNNDK
SEQ ID NO.19:NVIIHNIYFHDINAQPGALVK
SEQ ID NO.20:ALADCAQGFAKGTIGGK
SEQ ID NO.21:ALADCAQGFAK
SEQ ID NO.22:VEIINAGFR
SEQ ID NO.23:AFDGLVDAK
SEQ ID NO.24:FTDHVDQR
SEQ ID NO.25:HGFFQVVNNNYDR
SEQ ID NO.26:WGSYCIGGSAAPTILSQGNR
SEQ ID NO.27:FFAPDDPIK
SEQ ID NO.28:HGEAAQESMK
SEQ ID NO.29:FGAAQNRPLWIIFAR
Amb t 1.05’致敏蛋白质谱鉴定肽段:
SEQ ID NO.30:NVIIHNLNIHDVR
SEQ ID NO.31:SSGGPAIPR
SEQ ID NO.32:QDKAILLGASDTHFQDVK
SEQ ID NO.33:AILLGASDTHFQDVK
SEQ ID NO.34:FGFTQIVNNFYDR
SEQ ID NO.35:FVAPDNIYR GAKVELVYGGITLMNVK
SEQ ID NO.36:VELVYGGITLMNVK
SEQ ID NO.37:MHVTLAYNIFANTVHE
SEQ ID NO.38:WDKYAIGGSSNPTILSQGNK
SEQ ID NO.39:YAIGGSSNPTILSQGNK
SEQ ID NO.40:TGAQEPEWMTWNWR
SEQ ID NO.41:CKPDWAENR
SEQ ID NO.42:SLQGCEAHNIIDK
SEQ ID NO.43:RSLQGCEAHNIIDK
Amb t 8致敏蛋白质谱鉴定肽段:
SEQ ID NO.44:STSFPELKPDEINAIIK
SEQ ID NO.45:EFSEPGTLAPIGLFLAGAK
Amb t 10致敏蛋白质谱鉴定肽段:
SEQ ID NO.46:LGENYTVER
SEQ ID NO.47:SVDSDGDGFVDFEEFRK
Amb t 11致敏蛋白质谱鉴定肽段:
SEQ ID NO.48:ELESDEGFLGMYDR
SEQ ID NO.49:IPDKLDWR
SEQ ID NO.50:HGGIAPEANYPYVGK
SEQ ID NO.51:QNVPGLDEELLR
SEQ ID NO.52:APKDDPDLPK
Amb t 12致敏蛋白质谱鉴定肽段:
SEQ ID NO.53:TYDLNFKEENNDGKEK
SEQ ID NO.54:ISGEQLKDLYK
SEQ ID NO.55:ISGEQLK
表6豚草花粉致敏蛋白分子量、序列、过敏血清阳性率情况概览
5、理化性质检测
该豚草变应原浸液经理化性质检验后,其质量标准如表7:
表7豚草花粉变应原浸液理化性质质量标准
6、总变应原活性测定
当变应原浸液包含治疗有效量或诊断有效量的豚草花粉变应原时,用竞争性ELISA法测定相对生物效价为50000BAU/ml-200000BAU/ml。
7、无菌检查
不得有菌生长。
实施例7豚草花粉变应原浸液成品稳定性试验
对上述制备的豚草花粉标准化变应原浸液成品在2-8℃下进行长期稳定性研究试验,分别对三个批次样品分别检测0时,3个月,6个月,9个月和12个月的pH值变化,苯酚含量,甘油含量,氯化钠含量,蛋白含量和总变应原活性,主要致敏蛋白含量的变化情况,结果如图6至图11所示。
从上述稳定性结果中,可以发现在12个月长期试验中,豚草花粉标准化变应原浸液各质控参数虽有不规则波动,可能是实验误差导致,但均在质量标准质控范围内,且不同批次间变化趋势基本一致,这说明按照本发明所述的豚草花粉标准化变应原浸液,在2-8℃条件下,至少12月内能稳定地保存。因此,豚草花粉变应原浸液成品的效期合理预计在36个月。
实施例8本发明豚草变应原浸液与豚草变应原注射液医疗机构制剂(北京协和医院)品种稳定性Western Blotting对比研究
本发明变应原浸液组方中加入了50%的甘油作为稳定剂,优化了磷酸盐缓冲液pH及配方,使其更适合花粉变应原的浸提及保存,且原料经DNA条码技术鉴定,保障了纯度,原液采用了优化的冻干工艺,上述设计及工艺实现使按发明获得的变应原浸液成品稳定好、变应原活性得到良好保存,从而提高临床使用的有效性和安全性。为验证本发明下的处方及工艺对豚草变应原活性保存优于已有工艺及处方配制的同品种豚草变应原粗提液,本实施例针对豚草变应原浸液(本发明,即下述为A的产品)及豚草变应原注射液医疗机构制剂(北京协和医院配制,采用pH为8.2的Coca’s液提取,主要成分0.5%的NaCl、0.275%NaHCO3、0.4%苯酚和注射用水,即下述为B的产品)做了稳定性对比研究。
本实施例通过对比A产品和B产品4℃(温度控制2~6℃,避光保存)长期放置12月后,分别在0点和12月取样,以产品与豚草过敏患者血清池(15例豚草过敏患者组成的血清池,sIgE≥3级,皮试≥+++,典型的夏秋季花粉症病史者入选)的Western Blotting反应评价稳定性差异。
试验采用还原型SDS-PAG电泳,分离胶浓度为4~12%,0.2μm PVDF ImmobilonR-PSQ转印膜,一抗血清反应稀释度1:3,室温杂交2h,随后置于4℃冰箱杂交过夜,1×PBST洗3次,10min/次。。二抗Ms mAb to Hu IgE(ab99806)1:1000室温杂交2h,1×PBST洗3次,10min/次。将ECL显影液1:1混匀后曝光显色。
结果见图13所示,其中,M为蛋白分子量Marker(Genstar),N为浸液与健康受试者血清反应条带,L1和L2分别为本发明产品0时(A0)和12月(A12)与血清池反应结果,L3和L4分别为原医院制剂0时(B0)和12月(B12)与临床血清池反应条带结果。结果表明,本发明产品比原医院制剂产品在4℃放置12月后,不仅主要致敏蛋白条带清晰,其余多个致敏条带反应仍较明显,说明本发明产品中豚草致敏蛋白更为丰富,保存较好,活性也较高从而证实本发明产品更为稳定、有效。
实施例9评价应用1
目的:通过皮肤点刺试验(skin prick test,SPT)评价豚草花粉变应原浸液诊断豚草花粉过敏症的有效性和安全性。
方法如下:
选取2015年8月10日至2016年10月20日在北京协和医院变态(过敏)反应科就诊,患有过敏性鼻炎、过敏性哮喘、过敏性结膜炎、特应性皮炎、荨麻疹等过敏性疾病的门诊患者。对所有受试者进行协和豚草花粉变应原SPT,15min后测定平均风团直径(mean whealdiameter,MWD)。以豚草花粉血清特异性免疫球蛋白E specific immunoglobulin E,sIgE)作为确认豚草花粉过敏症的诊断标准,进行受试者工作特征曲线(receiver operatingcharacteristic curve,ROC曲线)分析,判断不同诊断界值下,本发明制备的豚草花粉变应原浸液临床诊断豚草花粉过敏症的准确性;同时记录不良事件,评价豚草花粉变应原浸液的安全性。结果本研究共计入组1026例,无脱落病例。全分析集(full analysis set,FAS)993例,平均年龄(31.28±14.55)岁。FAS的ROC曲线下面积(area under curve,AUC)为0.667(95%可信区间0.632~0.702)。据FAS的ROC估算SPT诊断豚草花粉过敏的MWD最佳诊断界值为3.75mm,特异度达95%时的MWD诊断界值为6.75mm。分别以MWD3.00、4.75和6.75mm为诊断界值,豚草花粉变应原浸液SPT诊断豚草花粉过敏的敏感度依次降低,分别为0.8273(95%可信区间0.7957~0.8589)、0.6200(95%可信区间0.5794~0.6606)、0.1673(95%可信区间0.1361~0.1985);而特异度依次升高,分别为0.3373(95%可信区间0.2789~0.3957)、0.6190(95%可信区间0.5591~0.6790)、0.9484(95%可信区间0.9211~0.9757)。安全集(save set,SS)1029例,6例出现7次不良事件,不良事件发生率0.58%(6/1029),主要表现为SPT后鼻痒、喷嚏、流涕、鼻塞、眼痒以及点刺部位局部皮肤出现较大风团反应。所有受试者均未出现严重不良事件。结论豚草花粉变应原浸液对于豚草花粉过敏症具有良好的诊断价值,安全性好。结合病史和不同的SPT诊断界值,可以提高诊断的准确度。
结果表明:本发明通过使用豚草花粉变应原浸液按1:1000稀释液做皮内试验分别与变态反应专科医生临床综合特异性诊断和血清特异性IgE(specific IgE,sIgE)诊断进行对比,皮内试验结果与临床综合特异性诊断及与血清sIgE诊断结果一致、具有较好的灵敏度和特异度,安全性好。
实施例10评价应用2
本发明的制备的豚草花粉变应原浸液对豚草花粉过敏患者全血进行嗜碱性粒细胞活化试验,能进行临床特异性过敏体外诊断。这个检测方法在IgE或非IgE介导的过敏反应中均适用,可以用于部分sIgE体外诊断存在假阴性、假阳性患者的确诊及部分过敏性休克患者不适于进行皮试诊断的情况。
试验原理:变应原与患者全血细胞反应可以模拟人体内变态反应过程:即特异性IgE抗体通过与相应的变应原桥联结合到细胞表面,激活细胞内信号级联导致嗜碱性粒细胞(CCR3持续表达于嗜碱性粒细胞,是其特异性标记)的活化脱颗粒。在这个脱颗粒的过程中,细胞内复合物影响跨膜蛋白CD63(gp53),使其外表达于细胞表面,并暴露于细胞外基质中,因此可以依赖流式细胞术原理(使用抗人趋化因子受体CCR3-藻红蛋白(anti-CCR3-PE)对嗜碱性粒细胞进行标记,使用抗人CD63单克隆抗体-异硫氰酸荧光素(anti-CD63-FITC)对活化状态的嗜碱性粒细胞进行标记,非特异性细胞活化剂fMLP作为一种阳性质控),并以嗜碱性粒细胞脱颗粒的百分数变化来判断受试者是否对特定变应原过敏。方法:选择健康受试者、豚草过敏患者,取其EDTA抗凝全血样本,以刺激缓冲液(阴性对照)、豚草变应原浸液(对1:103~1010稀释比例进行优化,本实施例中取1:108)、fMLP刺激液(阳性质控)作为嗜碱性粒细胞的激活物,加入到全血中,然后加入anti-CD63-FITC、anti-CCR3-PE染色,48h内上流式细胞仪进行检测。结果如图14所示,结果表明:健康受试者以阴性对照、豚草变应原浸液、阳性对照为嗜碱性粒细胞活化物时,其嗜碱性粒细胞活化率分别应<15%、<15%、≥15%,豚草花粉过敏患者以阴性对照、豚草变应原浸液、阳性对照为嗜碱性粒细胞活化物时,其嗜碱性粒细胞活化率分别应<15%、≥15%、≥15%。结论:该豚草变应原浸液能有效作为活化物运用于嗜碱性粒细胞活化试验,并依据其判断标准有效做出临床诊断。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院北京协和医院
<120> 一种豚草花粉变应原浸提物、其浸液及其制备方法
<150> 2018102437497
<151> 2019-03-23
<160> 55
<170> SIPOSequenceListing 1.0
<210> 38
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ggaagtaaaa gtcgtaacaa gg 22
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tcctccgctt attgatatgc 20
<210> 40
<211> 729
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gggttaaggc acgggtgtcg ctgactgggg tcgcgatcga agcatcatcg caagacaacg 60
cgttagggta ctttaagagt atctacaaca aggaatgaaa acgcacgaca cgagacgact 120
gtgttatcaa ccaccactag ccgtgcgtcc ctcttgatga gactcccatt taggccaacc 180
acaccatggg cacgggagac caatctccgc cccaaccaaa gtagtccctt gatgggaatg 240
tttggtgggg gcgacgtgat gcgtgacgcc caggcagacg tgccctcaac cggatggctt 300
cgggcgcaac ttgcgttcaa aaactcgatg gttcacggga ttctgcaatt cacaccaagt 360
atcgcatttt gctacgttct tcatcgatgc gtgagccgag atatccgttg ccgagagtcg 420
ttttaggttt aacaaagatg ccacatacaa cacgcacacc gcgaacgggg cgcaatagca 480
cgggcccttt aagtttagtt ttccttggca cgtaccgtgc cgggggtttg ttatcgtgtc 540
tccatgaaat ccatgatgcc ccaaatgttt ggcgcaagca tagacacacg tcgacaaggc 600
ctcacaaaga tcgaaacgta agcttcggtc ccagcttagc cggtgttttt acatgttcac 660
gggtcgttct gctatgcagg gttcgacaat gatccttccg caggttcacc tacggaaacc 720
ttgttacct 729
<210> 5
<211> 388
<212> PRT
<213> amino acid
<400> 5
Cys Cys Tyr Ile Leu Tyr Phe Thr Leu Ala Leu Val Ile Leu Leu Gln
1 5 10 15
Pro Val Arg Ser Ala Glu Asp Leu Gln Gln Ile Leu Pro Ser Val Asn
20 25 30
Glu Thr Arg Ser Leu Thr Thr Cys Glu Ala Asn Asn Ile Ile Asp Lys
35 40 45
Cys Trp Arg Cys Lys Pro Asp Trp Ala Glu Asn Arg Lys Ala Leu Ala
50 55 60
Asp Cys Ala Gln Gly Phe Ala Lys Gly Thr Ile Gly Gly Lys Met Gly
65 70 75 80
Asp Ile Tyr Thr Val Thr Ser Asp Leu Asp Asp Asp Val Ala Asn Pro
85 90 95
Lys Glu Gly Thr Leu Arg Phe Gly Ala Ala Gln Asn Arg Pro Leu Trp
100 105 110
Ile Ile Phe Ala Arg Asp Met Val Ile Arg Leu Asp Lys Glu Met Glu
115 120 125
Val Asn Asn Asp Lys Thr Ile Asp Gly Arg Gly Ala Arg Val Glu Ile
130 135 140
Ile Asn Ala Gly Phe Arg Ile Lys Glu Val Lys Asn Val Ile Ile His
145 150 155 160
Asn Ile Tyr Phe His Asp Ile Asn Ala Gln Pro Gly Ala Leu Val Lys
165 170 175
Arg Asn Glu Gly Pro Pro Arg Leu Arg Lys Gly Ser Asp Gly Asp Ala
180 185 190
Ile Ser Leu Asp Gly Cys Ser Gln Val Trp Ile Asp His Cys Ser Leu
195 200 205
Ser Lys Ala Phe Asp Gly Leu Val Asp Ala Lys Leu Gly Thr Thr Arg
210 215 220
Phe Thr Val Ser Asn Ser Leu Phe Thr Gln His Gln Tyr Val Leu Leu
225 230 235 240
Phe Trp Asp Phe Glu Glu Thr Gly Met Leu Gly Thr Val Ala Phe Asn
245 250 255
Lys Phe Thr Asp His Val Asp Gln Arg Met Pro Asn Cys Arg His Gly
260 265 270
Phe Phe Gln Val Val Asn Asn Asn Tyr Asp Arg Trp Gly Ser Tyr Cys
275 280 285
Ile Gly Gly Ser Ala Ala Pro Thr Ile Leu Ser Gln Gly Asn Arg Phe
290 295 300
Phe Ala Pro Asp Asp Pro Ile Lys Lys Asn Ala Val Gly Arg His Gly
305 310 315 320
Glu Ala Ala Gln Glu Ser Met Lys Trp Asn Trp Arg Ser Tyr Asn Asp
325 330 335
Leu Leu Glu Asn Gly Ala Ile Phe Val Ala Ser Gly Val Asp Pro Val
340 345 350
Leu Thr Pro Glu Gln Ser Glu Gly Met Ile Leu Ala Glu Pro Gly Glu
355 360 365
Ala Val Leu Arg Leu Thr Ser Ser Ala Gly Val Leu Ser Cys Gln Pro
370 375 380
Gly Ala Pro Cys
385
<210> 5
<211> 397
<212> PRT
<213> amino acid
<400> 5
Met Gly Ile Lys His Cys Cys Tyr Ile Leu Tyr Phe Thr Leu Ala Leu
1 5 10 15
Val Thr Leu Val Gln Ala Gly Arg Leu Gly Glu Glu Val Asp Ile Leu
20 25 30
Pro Ser Pro Asn Asp Thr Arg Arg Ser Leu Gln Gly Cys Glu Ala His
35 40 45
Asn Ile Ile Asp Lys Cys Trp Arg Cys Lys Pro Asp Trp Ala Glu Asn
50 55 60
Arg Lys Ala Leu Gly Asp Cys Ala Lys Gly Phe Gly Lys Gly Thr Tyr
65 70 75 80
Gly Gly Lys Gly Gly Asp Ile Tyr Met Val Thr Ser Asp Gln Asp Asp
85 90 95
Asp Val Val Asn Pro Arg Glu Gly Thr Leu Arg Phe Gly Ala Thr Gln
100 105 110
Asp Arg Pro Leu Trp Ile Ile Phe Glu Arg Asp Met Ile Ile Tyr Leu
115 120 125
Gln Gln Glu Met Val Val Thr Ser Asp Thr Thr Ile Asp Gly Arg Gly
130 135 140
Ala Lys Val Glu Leu Val Tyr Gly Gly Ile Thr Leu Met Asn Val Lys
145 150 155 160
Asn Val Ile Ile His Asn Leu Asn Ile His Asp Val Arg Val Leu Pro
165 170 175
Gly Gly Leu Ile Lys Ser Ser Gly Gly Pro Ala Ile Pro Arg Tyr Lys
180 185 190
Ser Asp Gly Asp Ala Val His Val Thr Gly Ser Ser Asp Ile Trp Ile
195 200 205
Asp His Cys Thr Leu Gly Asn Ser Phe Asp Gly Leu Val Asp Val Asn
210 215 220
Trp Gly Ser Ser Ser Val Thr Ile Ser Asn Cys Lys Phe Glu Arg Gln
225 230 235 240
Asp Lys Ala Ile Leu Leu Gly Ala Ser Asp Thr His Phe Gln Asp Val
245 250 255
Lys Met His Val Thr Leu Ala Tyr Asn Ile Phe Ala Asn Thr Val His
260 265 270
Glu Arg Met Pro Arg Cys Arg Phe Gly Phe Thr Gln Ile Val Asn Asn
275 280 285
Phe Tyr Asp Arg Trp Asp Lys Tyr Ala Ile Gly Gly Ser Ser Asn Pro
290 295 300
Thr Ile Leu Ser Gln Gly Asn Lys Phe Val Ala Pro Asp Asn Ile Tyr
305 310 315 320
Arg Lys Asn Val Cys Leu Arg Thr Gly Ala Gln Glu Pro Glu Trp Met
325 330 335
Thr Trp Asn Trp Arg Thr Gln Asn Asp Leu Leu Glu Asn Gly Ala Val
340 345 350
Phe Val Ala Ser Gly Ser Asp Pro Val Leu Thr Ala Glu Gln Asn Ala
355 360 365
Gly Met Ile Gln Ala Glu Pro Gly Glu Thr Val Pro Gln Leu Thr Met
370 375 380
Asn Ala Gly Val Leu Thr Cys Ser Pro Gly Ala Pro Cys
385 390 395
<210> 6
<211> 133
<212> PRT
<213> amino acid
<400> 6
Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Glu
1 5 10 15
Gly Thr Gly Gln His Leu Ala Ser Ala Ala Ile Phe Gly Thr Asp Gly
20 25 30
Asn Val Trp Ala Lys Ser Thr Ser Phe Pro Glu Leu Lys Pro Asp Glu
35 40 45
Ile Asn Ala Ile Ile Lys Glu Phe Ser Glu Pro Gly Thr Leu Ala Pro
50 55 60
Ile Gly Leu Phe Leu Ala Gly Ala Lys Tyr Met Val Ile Gln Gly Glu
65 70 75 80
Ser Gly Ala Val Ile Arg Gly Lys Lys Gly Ala Gly Gly Ile Cys Ile
85 90 95
Lys Lys Thr Gly Gln Ala Met Val Phe Gly Ile Tyr Glu Glu Pro Val
100 105 110
Asn Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu
115 120 125
Val Asp Gln Gly Met
130
<210> 7
<211> 160
<212> PRT
<213> amino acid
<400> 7
Met Ala Pro Glu Asn Asn Lys Leu Ser Ile Phe Pro Thr Asp Lys Glu
1 5 10 15
Glu Ile Thr Lys Ile Phe Asn Arg Phe Asp Thr Asn Gly Asp Gly Gln
20 25 30
Ile Ser Lys Glu Glu Leu Phe Asp Ile Leu Lys Ser Leu Gly Ser Asp
35 40 45
Thr Ser Pro Asp Glu Val Lys Arg Val Met Ala Glu Ile Asp Ala Asp
50 55 60
Gly Asp Gly Phe Ile Ser Leu Asp Glu Phe Val Leu Phe Cys Lys Gly
65 70 75 80
Met Glu Ala Glu Gly Asp Glu Met Asn Asp Leu Lys Glu Ala Phe Lys
85 90 95
Leu Tyr Asp Gln Asn Asn Asn Gly Val Ile Ser Ala Asn Glu Leu His
100 105 110
Gln Ile Leu Gly Arg Leu Gly Glu Asn Tyr Thr Val Glu Arg Cys Ala
115 120 125
Asp Met Ile Lys Ser Val Asp Ser Asp Gly Asp Gly Phe Val Asp Phe
130 135 140
Glu Glu Phe Arg Lys Met Met Ser Arg Lys Gly Gly Asp Gly Ala His
145 150 155 160
<210> 8
<211> 387
<212> PRT
<213> amino acid
<400> 8
Met Glu Ile Asn Lys Phe Val Ser Phe Ser Phe Ser Leu Val Leu Ile
1 5 10 15
Leu Gly Leu Ala Glu Ser Phe His Tyr His Glu Arg Glu Leu Glu Ser
20 25 30
Asp Glu Gly Phe Leu Gly Met Tyr Asp Arg Trp Arg Glu Gln His Asn
35 40 45
Ile Glu Met Arg Ser Pro Glu Arg Phe Asn Val Phe Lys Tyr Asn Val
50 55 60
Lys Arg Ile His Glu Ser Asn Lys Met Asp Lys Pro Tyr Lys Leu Lys
65 70 75 80
Val Asn Glu Phe Ala Asp Met Thr Asn Leu Glu Phe Val Asn Thr Tyr
85 90 95
Ala Asn Ser Lys Ile Ser His Phe Gln Ala Leu Arg Gly Gly Ala Pro
100 105 110
Gly Ser Leu Asp Thr Asp Pro Asn Lys Asp Phe Ile Tyr Ala Asn Ala
115 120 125
Thr Lys Ile Pro Asp Lys Leu Asp Trp Arg Glu Lys Asn Ala Val Thr
130 135 140
Asp Val Lys Gly Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ala Ala
145 150 155 160
Val Val Ala Leu Glu Gly Ile Asn Ala Ile Arg Thr Gly Lys Leu Val
165 170 175
Lys Phe Ser Glu Gln Gln Leu Val Asp Cys Asp Met Thr Asn Ala Gly
180 185 190
Cys Asp Gly Gly Leu Met Glu Pro Ala Phe Thr Tyr Val Ile Lys His
195 200 205
Gly Gly Ile Ala Pro Glu Ala Asn Tyr Pro Tyr Val Gly Lys Arg Glu
210 215 220
Thr Cys Asp Lys Ala Lys Ile Lys Asp Val Leu Lys Ile Asp Gly Arg
225 230 235 240
Gln Asn Val Pro Gly Leu Asp Glu Glu Leu Leu Arg Lys Ala Val Ala
245 250 255
His Gln Pro Val Ala Thr Gly Ile Gln Leu Ser Gly His Gly Leu Gln
260 265 270
Phe Tyr Ser Glu Gly Val Tyr Thr Gly Asp Cys Gly Thr Glu Pro Asn
275 280 285
His Gly Val Gly Ile Val Gly Tyr Gly Glu Asn Asp Lys Gly Ile Lys
290 295 300
Phe Trp Thr Val Lys Asn Ser Trp Gly Pro Thr Trp Gly Glu Lys Gly
305 310 315 320
Tyr Ile His Leu Gln Arg Gly Ala Arg Lys Glu Gly Leu Cys Gly Ile
325 330 335
Ala Met His Ser Ser Phe Pro Ile Met Asn Asp Pro Asn Pro Pro Lys
340 345 350
Asp Asp Pro Ile Lys Ala Pro Lys Asp Asp Pro Asp Leu Pro Lys Asp
355 360 365
Pro Lys Phe Lys Thr Thr Gln Arg Leu Gln Gly Ile Arg Thr Lys Leu
370 375 380
Leu Glu Leu
385
<210> 9
<211> 445
<212> PRT
<213> amino acid
<400> 9
Met Ala Thr Ile Lys Ala Val Lys Ala Arg Gln Ile Phe Asp Ser Arg
1 5 10 15
Gly Asn Pro Thr Val Glu Val Asp Val Thr Leu Ser Asp Gly Thr Leu
20 25 30
Ala Arg Ala Ala Val Pro Ser Gly Ala Ser Thr Gly Ile Tyr Glu Ala
35 40 45
Leu Glu Leu Arg Asp Gly Gly Ser Asp Tyr Leu Gly Lys Gly Val Leu
50 55 60
Lys Ala Val Asp Asn Val Asn Ser Ile Ile Gly Pro Ala Leu Ile Gly
65 70 75 80
Lys Asn Pro Thr Glu Gln Thr Lys Ile Asp Asn Phe Met Val Gln Glu
85 90 95
Leu Asp Gly Thr Val Asn Glu Trp Gly Trp Cys Lys Gln Lys Leu Gly
100 105 110
Ala Asn Ala Ile Leu Ala Val Ser Leu Ala Val Cys Lys Ala Gly Ala
115 120 125
Ser Val Lys Lys Ile Pro Leu Tyr Lys His Ile Ala Asn Leu Ala Gly
130 135 140
Asn Lys Thr Leu Val Leu Pro Val Pro Ala Phe Asn Val Ile Asn Gly
145 150 155 160
Gly Ser His Ala Gly Asn Lys Leu Ala Met Gln Glu Phe Met Ile Leu
165 170 175
Pro Val Gly Ala Ser Ser Phe Lys Glu Ala Met Lys Met Gly Val Glu
180 185 190
Val Tyr His Thr Leu Lys Ser Val Ile Lys Lys Lys Tyr Gly Gln Asp
195 200 205
Ala Thr Asn Val Gly Asp Glu Gly Gly Phe Ala Pro Asn Ile Gln Glu
210 215 220
Asn Lys Glu Gly Leu Glu Leu Leu Lys Thr Ala Ile Ser Lys Ala Gly
225 230 235 240
Tyr Ser Gly Lys Val Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe
245 250 255
Tyr Gly Glu Lys Asp Lys Thr Tyr Asp Leu Asn Phe Lys Glu Glu Asn
260 265 270
Asn Asp Gly Lys Glu Lys Ile Ser Gly Glu Gln Leu Lys Asp Leu Tyr
275 280 285
Lys Ser Phe Val Ser Glu Tyr Pro Ile Val Ser Ile Glu Asp Pro Phe
290 295 300
Asp Gln Asp Asp Trp Glu His Tyr Ala Lys Met Thr Ala Glu Cys Gly
305 310 315 320
Glu Gln Val Gln Ile Val Gly Asp Asp Leu Leu Val Thr Asn Pro Thr
325 330 335
Arg Val Lys Lys Ala Ile Asp Glu Lys Thr Cys Asn Ala Leu Leu Leu
340 345 350
Lys Val Asn Gln Ile Gly Ser Val Thr Glu Ser Ile Glu Ala Val Arg
355 360 365
Met Ser Lys His Ala Gly Trp Gly Val Met Ala Ser His Arg Ser Gly
370 375 380
Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu Ser Val Gly Leu Ala Thr
385 390 395 400
Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg Ser Glu Arg Leu Ala Lys
405 410 415
Tyr Asn Gln Leu Leu Arg Ile Glu Glu Glu Leu Gly Ser Glu Ala Val
420 425 430
Tyr Ala Gly Ala Asn Phe Arg Lys Pro Val Glu Pro Tyr
435 440 445
<210> 10
<211> 1167
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgttgttaca tcttgtattt taccttagcc cttgtcattt tgctgcaacc tgttcgttct 60
gccgaagatc tccagcaaat cttaccttca gttaacgaaa caaggagcct gacaacatgt 120
gaagcaaaca acattataga caagtgctgg aggtgcaaac ctgattgggc agaaaaccga 180
aaagcgttag ccgattgtgc ccaaggtttt gcaaagggaa ccatcggcgg aaaaatgggt 240
gatatctaca cggtgaccag tgatctagat gatgatgttg ctaatccaaa agaaggcaca 300
ctccggtttg gtgccgccca aaacaggccc ttgtggatta tttttgcaag agatatggtg 360
attcgtttgg ataaagagat ggaggtaaac aacgacaaga ccatcgatgg ccgaggggcg 420
agagttgaaa tcattaacgc tggtttccgc atcaaagaag tcaagaatgt aatcattcat 480
aacatatatt ttcatgatat taatgcgcag ccaggagccc tggttaagag aaacgaaggt 540
ccaccacgtc taagaaaggg tagtgatggt gatgctataa gtcttgatgg ttgttcacaa 600
gtatggatcg accattgctc gctcagtaag gcttttgatg ggctagtcga tgccaaactc 660
ggcaccacac gcttcaccgt ttccaacagc ttatttaccc aacaccaata tgtattattg 720
ttttgggatt ttgaagaaac aggcatgcta ggaacggtcg cattcaacaa gttcactgat 780
cacgttgacc aaagaatgcc taattgtcgg catgggtttt tccaagtcgt taacaacaac 840
tacgatagat ggggatcgta ttgcatcggt ggtagcgcgg ccccaaccat actcagccaa 900
ggaaacagat tctttgcccc cgatgatcct atcaagaaaa atgctgtagg gaggcatggt 960
gaagccgcac aagagtcgat gaagtggaac tggagatcgt ataatgatct gcttgaaaat 1020
ggtgctattt ttgttgcatc cggcgttgat ccagtgttaa cccctgaaca aagcgaaggg 1080
atgattctag ccgaaccagg tgaagccgtt ctaagactca ctagtagtgc cggtgtactc 1140
tcatgccaac ctggagcacc ttgctaa 1167
<210> 11
<211> 1194
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggggatca aacattgttg ttacatcttg tattttacct tagcacttgt cactttggtg 60
caagctggac gtcttggcga agaggtcgac atcttacctt cacctaacga tacaaggagg 120
agcctgcaag gatgtgaagc acacaacatt atagacaagt gttggaggtg caaacccgat 180
tgggcggaga accgaaaagc gttaggcgat tgtgccaaag gttttggaaa gggaacttac 240
ggcggaaaag ggggtgatat ctacatggtc acaagtgatc aggatgatga tgttgtgaat 300
ccaagagaag gcacactccg gttcggtgcc acccaagaca ggcccttgtg gatcattttt 360
gaaagagata tgattattta tttgcaacaa gagatggtag taaccagcga cacgaccatc 420
gatggtcgag gggcgaaagt tgagcttgtt tatggaggta tcaccctcat gaatgtcaag 480
aatgtaatca ttcacaacct aaatatccat gatgttagag tgcttccagg aggcctgatt 540
aagtccagcg gtggtccagc aataccaaga tataagagtg atggtgatgc tgtccatgtt 600
actggtagtt cagacatatg gatcgaccat tgcacgctcg gcaattcatt tgatgggctc 660
gtcgatgtca actggggtag ctcatcagta accatttcca actgcaaatt cgaacgccaa 720
gacaaagcca ttttgctcgg ggctagtgac acccattttc aagatgtgaa aatgcatgta 780
acgcttgcat acaacatctt cgccaatacc gttcatgaaa gaatgcctag atgccgattt 840
gggtttaccc aaattgtaaa caacttctac gacagatggg ataagtacgc cattggtggt 900
agctcgaacc ctactattct cagccaaggg aacaaattcg tggcccccga taacatttac 960
aggaaaaacg tctgtctgag gactggtgca caggagccag aatggatgac ttggaactgg 1020
agaacgcaaa acgacctgct tgaaaatggt gctgtctttg tggcatccgg gtctgatcca 1080
gtgctaaccg ctgaacaaaa tgcaggcatg attcaagctg aacctggaga aacggttccg 1140
caactcacca tgaatgctgg tgtactcaca tgctcgcctg gagcaccttg ctaa 1194
<210> 12
<211> 402
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgtcgtggc agacttacgt ggatgaacat ttgatgtgtg acatcgaagg cactggtcag 60
catctcgcct ctgccgctat cttcggtacc gatggcaacg tctgggccaa gagcacttct 120
ttccccgagt tgaaacctga tgagattaat gctatcatca aagaattcag tgaacctggt 180
actcttgccc ctattggttt attcctagcc ggtgcaaagt acatggtgat ccaaggcgag 240
tctggagctg ttattcgtgg aaagaaggga gctggaggga tctgcatcaa gaaaactggt 300
caagccatgg tgtttggcat ctatgaggag ccggtgaatc ctggtcagtg caacatggtt 360
gtggaaaggt tgggtgatta tcttgtggat caaggcatgt aa 402
<210> 13
<211> 402
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgtcgtggc agacttacgt ggatgaacat ttgatgtgtg acatcgaagg cactggtcag 60
catctcgcct ctgccgctat cttcggtacc gatggcaacg tctgggccaa gagcacttct 120
ttccccgagt tgaaacctga tgagattaat gctatcatca aagaattcag tgaacctggt 180
actcttgccc ctattggttt attcctagcc ggtgcaaagt acatggtgat ccaaggcgag 240
tctggagctg ttattcgtgg aaagaaggga gctggaggga tctgcatcaa gaaaactggt 300
caagccatgg tgtttggcat ctatgaggag ccggtgaatc ctggtcagtg caacatggtt 360
gtggaaaggt tgggtgatta tcttgtggat caaggcatgt aa 402
<210> 14
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggaaatca acaagtttgt ttctttttca ttttctttgg ttttgatttt aggacttgcg 60
gagagcttcc attaccatga gagagagctc gaatcggacg aggggttttt ggggatgtac 120
gataggtgga gggagcaaca caatatcgaa atgagaagcc cagaacggtt caatgtattc 180
aaatacaatg ttaaacgcat tcacgaatcg aataagatgg acaagccgta taagttgaag 240
gtgaacgagt ttgctgacat gactaacctt gagtttgtta acacgtatgc taactcgaag 300
attagccatt ttcaagctct ccgaggagga gcacctggat cgcttgatac cgaccctaat 360
aaagatttca tatatgcaaa tgcgactaaa atcccagata agcttgattg gagagagaaa 420
aatgccgtca ctgatgtcaa gggtcaaggc ggatgtggaa gttgttgggc atttgccgct 480
gtggttgcat tggaaggaat aaacgcgatc agaaccggaa aactagtaaa attttccgaa 540
caacaactcg tcgactgtga catgacgaac gcaggatgcg acggagggct aatggaacct 600
gcattcacat acgtcataaa gcatggaggt atagcaccag aagccaacta cccttacgta 660
ggcaaaagag aaacctgcga caaagcaaag attaaagatg tgttgaagat cgatggtaga 720
caaaatgtgc ctggacttga tgaagaatta ctaaggaagg cagtagcaca ccagcctgta 780
gctacaggca tacaacttag cggccatggt ttgcagttct attccgaggg tgtatatacc 840
ggagattgtg gtacagagcc gaatcatgga gttggaattg tgggatacgg tgagaatgat 900
aaggggatta aattttggac cgtgaagaac tcatggggac caacgtgggg agagaaggga 960
tacatacatc tacaacgcgg agctaggaaa gagggactct gcggaatagc aatgcattct 1020
tcttttccta ttatgaacga cccaaatcca cctaaagacg accccattaa agcacctaaa 1080
gacgaccctg atttacctaa ggaccctaaa tttaaaacga ctcagaggtt gcaggggata 1140
aggactaaat tgttggagtt gtga 1164
<210> 15
<211> 1338
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggcgacga tcaaagccgt taaagctcgt cagatcttcg atagtcgtgg taatcctacg 60
gtcgaagttg atgttactct gtctgatgga actttggcta gagccgctgt acctagcggt 120
gcttctacag gtatatatga agcgttggag ttgcgagatg gaggttcgga ttatctcggt 180
aaaggtgtct tgaaggctgt ggacaatgtt aactcgatta ttggtcctgc cttgattggc 240
aagaatccta cggaacagac caaaattgac aacttcatgg ttcaagagct tgatggaact 300
gtaaatgaat ggggctggtg caaacagaag cttggtgcca atgccatact ggcagtgtca 360
cttgctgtgt gtaaagctgg agcatctgtt aagaaaattc ctctatataa gcacattgca 420
aacctagctg gaaacaagac actggtgttg ccagtaccag catttaatgt gatcaatggt 480
gggtcgcatg caggcaacaa acttgcaatg caagagttta tgattctccc tgttggagct 540
tcttccttca aggaagccat gaagatgggt gttgaagtgt atcacacctt gaagtctgta 600
attaaaaaga aatatggtca agatgctacc aatgttggtg atgaaggtgg atttgctcct 660
aacatccagg aaaacaaaga aggccttgaa cttttgaaga ctgcaatatc aaaagctgga 720
tacagcggaa aggttgttat tggaatggat gttgctgcat ctgaattcta tggcgagaag 780
gacaagacct atgatctgaa cttcaaggaa gagaacaacg atggcaaaga aaagatttca 840
ggagaacaac taaaagatct ttacaagtcg tttgtgagtg agtaccccat tgtgtccatt 900
gaggacccat ttgaccaaga tgactgggag cactatgcca agatgaccgc tgaatgtggc 960
gaacaagttc aaattgtagg agacgatctt ttggtcacca accccacgag agtcaagaag 1020
gcaattgatg agaagacttg caatgctctt cttctaaagg tcaaccaaat tgggtctgtg 1080
accgagagta tcgaagctgt gaggatgtcc aaacatgctg gttggggtgt gatggccagt 1140
caccgcagtg gagaaacaga ggacaccttc attgctgatc tttctgtggg tttggcaacg 1200
ggtcaaatca aaactggagc tccatgcagg tcagagcgtc ttgcaaagta caaccagctg 1260
ttgagaatcg aagaagagct tggatcagaa gcggtttatg ctggagccaa cttccgcaag 1320
ccagtggaac cctactag 1338
<210> 16
<211> 19
<212> PRT
<213> amino acid
<400> 16
Met Gly Asp Ile Tyr Thr Val Thr Ser Asp Leu Asp Asp Asp Val Ala
1 5 10 15
Asn Pro Lys
<210> 17
<211> 12
<212> PRT
<213> amino acid
<400> 17
Lys Ala Leu Ala Asp Cys Ala Gln Gly Phe Ala Lys
1 5 10
<210> 18
<211> 11
<212> PRT
<213> amino acid
<400> 18
Leu Asp Lys Glu Met Glu Val Asn Asn Asp Lys
1 5 10
<210> 19
<211> 21
<212> PRT
<213> amino acid
<400> 19
Asn Val Ile Ile His Asn Ile Tyr Phe His Asp Ile Asn Ala Gln Pro
1 5 10 15
Gly Ala Leu Val Lys
20
<210> 20
<211> 17
<212> PRT
<213> amino acid
<400> 20
Ala Leu Ala Asp Cys Ala Gln Gly Phe Ala Lys Gly Thr Ile Gly Gly
1 5 10 15
Lys
<210> 21
<211> 11
<212> PRT
<213> amino acid
<400> 21
Ala Leu Ala Asp Cys Ala Gln Gly Phe Ala Lys
1 5 10
<210> 22
<211> 9
<212> PRT
<213> amino acid
<400> 22
Val Glu Ile Ile Asn Ala Gly Phe Arg
1 5
<210> 23
<211> 9
<212> PRT
<213> amino acid
<400> 23
Ala Phe Asp Gly Leu Val Asp Ala Lys
1 5
<210> 24
<211> 8
<212> PRT
<213> amino acid
<400> 24
Phe Thr Asp His Val Asp Gln Arg
1 5
<210> 25
<211> 13
<212> PRT
<213> amino acid
<400> 25
His Gly Phe Phe Gln Val Val Asn Asn Asn Tyr Asp Arg
1 5 10
<210> 26
<211> 20
<212> PRT
<213> amino acid
<400> 26
Trp Gly Ser Tyr Cys Ile Gly Gly Ser Ala Ala Pro Thr Ile Leu Ser
1 5 10 15
Gln Gly Asn Arg
20
<210> 27
<211> 9
<212> PRT
<213> amino acid
<400> 27
Phe Phe Ala Pro Asp Asp Pro Ile Lys
1 5
<210> 28
<211> 10
<212> PRT
<213> amino acid
<400> 28
His Gly Glu Ala Ala Gln Glu Ser Met Lys
1 5 10
<210> 29
<211> 15
<212> PRT
<213> amino acid
<400> 29
Phe Gly Ala Ala Gln Asn Arg Pro Leu Trp Ile Ile Phe Ala Arg
1 5 10 15
<210> 30
<211> 13
<212> PRT
<213> amino acid
<400> 30
Asn Val Ile Ile His Asn Leu Asn Ile His Asp Val Arg
1 5 10
<210> 31
<211> 9
<212> PRT
<213> amino acid
<400> 31
Ser Ser Gly Gly Pro Ala Ile Pro Arg
1 5
<210> 32
<211> 18
<212> PRT
<213> amino acid
<400> 32
Gln Asp Lys Ala Ile Leu Leu Gly Ala Ser Asp Thr His Phe Gln Asp
1 5 10 15
Val Lys
<210> 33
<211> 15
<212> PRT
<213> amino acid
<400> 33
Ala Ile Leu Leu Gly Ala Ser Asp Thr His Phe Gln Asp Val Lys
1 5 10 15
<210> 34
<211> 13
<212> PRT
<213> amino acid
<400> 34
Phe Gly Phe Thr Gln Ile Val Asn Asn Phe Tyr Asp Arg
1 5 10
<210> 35
<211> 26
<212> PRT
<213> amino acid
<400> 35
Phe Val Ala Pro Asp Asn Ile Tyr Arg Gly Ala Lys Val Glu Leu Val
1 5 10 15
Tyr Gly Gly Ile Thr Leu Met Asn Val Lys
20 25
<210> 36
<211> 14
<212> PRT
<213> amino acid
<400> 36
Val Glu Leu Val Tyr Gly Gly Ile Thr Leu Met Asn Val Lys
1 5 10
<210> 37
<211> 16
<212> PRT
<213> amino acid
<400> 37
Met His Val Thr Leu Ala Tyr Asn Ile Phe Ala Asn Thr Val His Glu
1 5 10 15
<210> 38
<211> 20
<212> PRT
<213> amino acid
<400> 38
Trp Asp Lys Tyr Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser
1 5 10 15
Gln Gly Asn Lys
20
<210> 39
<211> 17
<212> PRT
<213> amino acid
<400> 39
Tyr Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Gln Gly Asn
1 5 10 15
Lys
<210> 40
<211> 14
<212> PRT
<213> amino acid
<400> 40
Thr Gly Ala Gln Glu Pro Glu Trp Met Thr Trp Asn Trp Arg
1 5 10
<210> 41
<211> 9
<212> PRT
<213> amino acid
<400> 41
Cys Lys Pro Asp Trp Ala Glu Asn Arg
1 5
<210> 42
<211> 13
<212> PRT
<213> amino acid
<400> 42
Ser Leu Gln Gly Cys Glu Ala His Asn Ile Ile Asp Lys
1 5 10
<210> 43
<211> 14
<212> PRT
<213> amino acid
<400> 43
Arg Ser Leu Gln Gly Cys Glu Ala His Asn Ile Ile Asp Lys
1 5 10
<210> 44
<211> 17
<212> PRT
<213> amino acid
<400> 44
Ser Thr Ser Phe Pro Glu Leu Lys Pro Asp Glu Ile Asn Ala Ile Ile
1 5 10 15
Lys
<210> 45
<211> 19
<212> PRT
<213> amino acid
<400> 45
Glu Phe Ser Glu Pro Gly Thr Leu Ala Pro Ile Gly Leu Phe Leu Ala
1 5 10 15
Gly Ala Lys
<210> 46
<211> 9
<212> PRT
<213> amino acid
<400> 46
Leu Gly Glu Asn Tyr Thr Val Glu Arg
1 5
<210> 47
<211> 17
<212> PRT
<213> amino acid
<400> 47
Ser Val Asp Ser Asp Gly Asp Gly Phe Val Asp Phe Glu Glu Phe Arg
1 5 10 15
Lys
<210> 48
<211> 14
<212> PRT
<213> amino acid
<400> 48
Glu Leu Glu Ser Asp Glu Gly Phe Leu Gly Met Tyr Asp Arg
1 5 10
<210> 49
<211> 8
<212> PRT
<213> amino acid
<400> 49
Ile Pro Asp Lys Leu Asp Trp Arg
1 5
<210> 50
<211> 15
<212> PRT
<213> amino acid
<400> 50
His Gly Gly Ile Ala Pro Glu Ala Asn Tyr Pro Tyr Val Gly Lys
1 5 10 15
<210> 51
<211> 12
<212> PRT
<213> amino acid
<400> 51
Gln Asn Val Pro Gly Leu Asp Glu Glu Leu Leu Arg
1 5 10
<210> 52
<211> 10
<212> PRT
<213> amino acid
<400> 52
Ala Pro Lys Asp Asp Pro Asp Leu Pro Lys
1 5 10
<210> 53
<211> 16
<212> PRT
<213> amino acid
<400> 53
Thr Tyr Asp Leu Asn Phe Lys Glu Glu Asn Asn Asp Gly Lys Glu Lys
1 5 10 15
<210> 54
<211> 11
<212> PRT
<213> amino acid
<400> 54
Ile Ser Gly Glu Gln Leu Lys Asp Leu Tyr Lys
1 5 10
<210> 55
<211> 7
<212> PRT
<213> amino acid
<400> 55
Ile Ser Gly Glu Gln Leu Lys
1 5