CN113354719A - 奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用 - Google Patents

奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用 Download PDF

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CN113354719A
CN113354719A CN202110512889.1A CN202110512889A CN113354719A CN 113354719 A CN113354719 A CN 113354719A CN 202110512889 A CN202110512889 A CN 202110512889A CN 113354719 A CN113354719 A CN 113354719A
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沈克飞
付利芝
杨睿
张素辉
徐登峰
郑华
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Chongqing Academy of Animal Sciences
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Abstract

本发明涉及生化领域，具体涉及奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用。本发明提供了一种变形杆菌的抗原鉴定、制备，及抗原在检测抗变形杆菌IgG抗体的间接ELISA方法中的可行性。该间接ELISA方法能有效区分奇异变形杆菌、普通变形杆菌与大肠杆菌、金黄色葡萄球菌、化脓隐秘杆菌、伪结核棒状杆菌感染，表明该抗原能用于检测、诊断奇异变形杆菌、普通变形杆菌感染。

Description

奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用
技术领域
本发明涉及生化领域，具体涉及奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用。
背景技术
变形杆菌(Proteus)属肠杆菌科变形杆菌属，革兰氏阴性菌。该菌广泛分布于自然界，多存在于污染的水、土壤、空气以及人或动物的排泄物之中。有关国外资料显示，变形杆菌属可分为5个已命名的种，即普通变形杆菌(Proteus vulgaris)、奇异变形杆菌(Proteusmirabilis)、粘化变形杆菌(Proteus myxofaciens)、潘氏变形杆菌(Proteus penneri)和豪氏变形杆菌(Proteus hauseri)以及3个未命名的种。国内资料则将变形杆菌属分为普通变形杆菌(P.vulgaris)、奇异变形杆菌(P.mirabilis)、产粘液变形杆菌(P.myxofaciens)和潘纳变形杆菌(P.penneri)4种，其中奇异变形杆菌和普通变形杆菌是常见的机会性致病菌和食源性病原体。
革兰氏阴性菌的抗原主要有O抗原、外膜蛋白、鞭毛、菌毛、荚膜等。变形杆菌根据其O抗原差异性可以划分为进行分化为至少80个O抗原血清型，但在常规诊断中不包括血清型分析。O抗原又称O-特异链(O-specific chain)或称O-多糖，是脂多糖的最外层部分，由数十个相同的寡糖单位组成。某些血清型变形杆菌血清与立克次体等病原体表现出抗原交叉反应。细菌外膜位于壳聚糖外侧，它包裹整个细菌，是革兰氏阴性菌的1种特有结构，由脂多糖、磷脂、蛋白质和脂蛋白等组成，厚度约8-10nm，约占细胞壁的80％。外膜蛋白是存在于细菌外膜中以及与外膜相关联的所有蛋白总称，是构成外膜的主要成分，它在物质运输与维持外膜结构等方面具有重要意义。外膜蛋白主要蛋白包括外膜蛋白A(OMPA)、微孔蛋白、脂蛋白等，其细胞内表达拷贝数约为1×105-2×105。外膜蛋白既是潜在毒力因子，更具有良好的免疫原性，已经证实OMPA是变形杆菌的1种保护性抗原，能够有效促进B淋巴细胞的分裂。
目前，在变形杆菌抗原方面的研究主要集中在O抗原及血清型分型上，在蛋白质抗原上的鉴定与应用上的研究报告不多。变形杆菌的检测与诊断方法有核酸检测、选择性培养基培养、生化鉴定和血清学检测等，常用的方法是核酸检测。在血清学检测方法上一是缺少对诊断用抗原的评价，二是缺少相应的可以应用的检测或诊断技术方法。为此，亟需一种变形杆菌的抗原鉴定、制备方法。
发明内容
本发明为了解决上述问题，提供了一种变形杆菌的抗原鉴定、制备，及抗原在检测抗变形杆菌IgG抗体的间接ELISA方法中的可行性。该间接ELISA方法能有效区分奇异变形杆菌、普通变形杆菌与大肠杆菌、金黄色葡萄球菌、化脓隐秘杆菌、伪结核棒状杆菌感染，表明该抗原能用于检测、诊断奇异变形杆菌、普通变形杆菌感染。
本发明目的之一在于提供一种奇异变形杆菌膜蛋白的膜外部分蛋白，所述膜外部分蛋白的氨基酸序列依次由SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8组成。
本发明目的之二在于提供一种包括优化后的上述膜外部分蛋白的重组质粒，所述重组质粒的核苷酸序列依次由SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12组成。
具体的，所述重组质粒的制备方法是依次将所述SEQ ID NO.9、SEQ ID NO.10、SEQID NO.11和SEQ ID NO.12的核苷酸序列克隆至pETET-28a中。
本发明目的之三在于提供上述技术方案中的重组质粒制备得到的基因工程菌株。
本发明目的之四在于提供一种利用上述方案中的基因工程菌株产生的重组蛋白。
本发明目的之五在于公开将上述的重组蛋白在制备检测奇异变形杆菌或普通变形杆菌的试剂盒中的应用。
本发明目的之六在于提供上述技术方案中的重组蛋白检测奇异变形杆菌或普通变形杆菌的方法，检测样本为奇异变形杆菌和/或普通变形杆菌和/或伪结核棒状杆菌和/或金黄色葡萄球菌和/或大肠杆菌和/或化脓隐秘杆菌，具体包括如下步骤：
(1)纯化所述重组蛋白；
(2)以rOMP包被所述纯化重组蛋白；
(3)利用间接ELISA方法检测血清的A450；
(4)当所述
Figure BDA0003060973170000031
时，检测为奇异变形杆菌或普通变形杆菌。
具体的，步骤(2)包被过程中加入HRP标记的山羊抗小鼠IgG抗体。
具体的，利用四甲基联苯胺显色10min，酶标仪测定A450值。
具体的，所述纯化重组蛋白的纯度为91％。
具体的，包被过程中所述纯化重组蛋白的浓度为0.27μg/mL。
具体的，其特征在于，所述
Figure BDA0003060973170000032
本发明目的之七在于提供上述技术方案中的重组蛋白将奇异变形杆菌、普通变形杆菌与伪结核棒状杆菌、金黄色葡萄球菌、大肠杆菌、化脓隐秘杆菌区分开来的方法，利用所述重组蛋白作为抗原，所述抗原作为捕获剂被固相载体固定，用于捕获特异性抗体，所述特异性抗体为HRP标记的山羊抗小鼠IgG抗体；通过间接ELISA方法检测所述山羊抗小鼠IgG抗体，测定A450区分出奇异变形杆菌和普通变形杆菌。
具体的当所述
Figure BDA0003060973170000033
时，检测为奇异变形杆菌或普通变形杆菌。
具体的，利用四甲基联苯胺显色10min，酶标仪测定A450值。
具体的，所述纯化重组蛋白的纯度为91％。
具体的，包被过程中所述纯化重组蛋白的浓度为0.27μg/mL。
具体的，其特征在于，所述
Figure BDA0003060973170000034
本发明的有益之处在于：本发明的重组蛋白抗原性较强，利用间接ELISA方法能有效区分奇异变形杆菌、普通变形杆菌与大肠杆菌、金黄色葡萄球菌、化脓隐秘杆菌、伪结核棒状杆菌感染，表明该抗原能用于检测、诊断奇异变形杆菌、普通变形杆菌感染。
附图说明
图1为本发明的提取的奇异变形杆菌膜蛋白
图2为本发明的奇异变形杆菌膜蛋白与抗血清反应
图3为OMP蛋白质跨膜结构域、信号肽预测图
图4为SH蛋白质跨膜结构域、信号肽预测图
图5为GPD蛋白质跨膜结构域、信号肽预测图
图6为SBP蛋白质跨膜结构域、信号肽预测图
图7为重组蛋白的诱导表达分析
图8为重组蛋白的反应原性分析
图9为rOMP的纯化
具体实施方式
下面通过实施例对本发明进行进一步详细说明，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，本领域技术人员应该理解的是，在不偏离本发明的结构思路、使用范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1操作方法
菌株：奇异变形杆菌、普通变性杆菌、大肠杆菌、金黄色葡萄球菌、化脓隐秘杆菌、伪结核棒状杆菌由重庆畜牧科学院兽医研究所保存和提供。
主要试剂：细菌膜蛋白分离试剂盒购自贝博公司；碱性磷酸酶标记的山羊抗小鼠IgG抗体、辣根过氧化物酶(HRP)标记的山羊抗小鼠IgG抗体购自EarthOx Life Sciences；Ni-NTA填料购自北京诺和生物技术有限公司；Quick Start Bradford购自伯乐生命医学产品(上海)有限公司；BCTP/NBT购自上海生工生物工程有限公司；TMB单组分显色液购自索莱宝生物科技有限公司；酶标板购自康宁生命科学(吴江)有限公司；明胶购自广州赛国生物科技有限公司；蛋白质预染Marker北京索莱宝科技有限公司。
实验动物：身体质量约为25g的雌性SPF昆明小鼠购自中国人民解放军第三军医大学。具体方法如下：
1、细菌培养：奇异变形杆菌、普通变形杆菌、金黄色葡萄球菌、大肠杆菌单菌落分别接种于LB培养基，37℃振摇培养过夜。化脓隐秘杆菌单菌落接种于含8％小牛血清的TSB培养基，37℃振摇培养48h。伪结核棒状杆菌单菌落接种于含8％小牛血清的TSB培养基，37℃振摇培养24h。
2、攻毒：将上述培养物以亚致死剂量接种小鼠，接种途径为腹腔注射。接种后15d采集小鼠血液，分离血清，即为抗血清。血清于-20℃保存。
3、膜蛋白分离提取：使用细菌膜蛋白分离试剂盒分离提取新鲜培养的奇异变形杆菌膜蛋白，具体操作按说明书要求进行。采用SDS-PAGE检测提取的膜蛋白，采用免疫印迹技术分析提取的膜蛋白与奇异变形杆菌攻毒的小鼠抗血清(1:100稀释)的反应原性。
4、质谱与序列分析：切下与小鼠抗血清反应的聚丙烯酰胺凝胶上对应的条带，采用LC-MS/MS方法对样品进行质谱分析以获取蛋白质序列信息。将获得的蛋白质进行跨膜结构域和信号肽分析(https://phobius.sbc.su.se/)，除去跨膜结构域及信号肽部分，选择膜外部分用于表达。
5、基因序列优化及表达系统构建：将用于表达的基因根据大肠杆菌密码子偏好性优化基因序列，在基因的5’和3’端分别加入BamHⅠ和XhoⅠ限制性内切酶识别位点。采用化学合成法合成基因，将其克隆至pETET-28a构建重组表达载体，转化到大肠杆菌BL21(DE3)菌株中。
6、重组蛋白的诱导表达与反应原性分析：37℃下振摇培养至菌体浓度达OD600约为1时，加入终浓度0.1mmol/L IPTG，37℃下诱导表达3h。培养物4℃下5 000×g离心10min，沉淀用Tris-HCl(pH8.0)洗涤3次后用Tris-HCl悬浮，冰水浴中超声裂解菌体(超声功率为300W，工作4s间歇5s，超声裂解15min)。取1mL菌体裂解液，12 000×g离心1min，上清和沉淀分别加入SDS-PAGE上样缓冲液，沸水浴10min，进行SDS-PAGE和免疫印迹分析。免疫印迹具体操作为：样品经SDS-PAGE后转印至硝酸纤维素膜上，采用5％脱脂奶粉封闭2h，TBST洗3次后与奇异变形杆菌攻毒的小鼠抗血清(1:100稀释)孵育1h，TBST洗3次后与碱性磷酸酶标记的山羊抗小鼠IgG抗体(1:3000稀释)孵育1h，TBST洗3次后置BCTP/NBT中显色。
7、重组蛋白质纯化：提取与奇异变形杆菌攻毒的小鼠抗血清有反应的重组蛋白。根据SDS-PAGE分析重组蛋白主要以包含体形式表达。12 000×g离心10min，从菌体裂解液中收集包含体沉淀，用含0.5mol/L NaCl、0.01mol/L EDTA和2％Triton X-100的0.05mol/LTris-HCl(pH8.0)洗涤后，加入8mol/L尿素以溶解包含体沉淀。12 000×g离心10min，收集上清，上清经0.22μm滤膜过滤后加入至处理好的Ni-NTA介质中以吸附重组蛋白，用含20mmol/L咪唑、0.5mol/L NaCl、8mol/L尿素的0.02mol/L Tris-HCl(pH8.0)洗杂蛋白，用含150mmol/L咪唑、0.5mol/L NaCl、8mol/L尿素的0.02mol/L Tris-HCl(pH8.0)洗脱结合的重组蛋白。纯化产物进行SDS-PAGE分析，用Image Lab3.0对目的条带进行灰度扫描以分析纯度。
8、包被抗原浓度的确定：用纯化的重组蛋白包被96孔板，抗原包被浓度为0.27、0.09和0.03μg/mL，4℃包被过夜；2％明胶封闭液于37℃封闭2h；加入血清(1:100稀释)，37℃反应30min；用PBST洗涤3次，加入HRP标记的山羊抗小鼠IgG抗体(1:20 000稀释)，37℃反应60min；四甲基联苯胺显色10min，用酶标仪测定A450值。以奇异变形杆菌攻毒的小鼠血清作为阳性血清，阳性血清3份，以未攻毒小鼠血清作为阴性血清，阴性血清3份，以酶标仪测得的阳性血清显色后450nm波长的吸光值(A450)值>1、且阳性血清A450均值与阴性血清A450均值比值(P/N值)最大的反应条件为最佳反应条件。
9、阴阳性临界值的确定：以未攻毒奇异变性杆菌的小鼠血清(包括大肠杆菌、金黄色葡萄球菌、化脓隐秘杆菌、伪结核棒状杆菌攻毒的小鼠血清及未攻毒小鼠血清，每种类别血清10份)作为阴性血清，采用优化的间接ELISA法测定50份阴性血清，计算阴性血清A450的平均值
Figure BDA0003060973170000062
和标准差(SD)，
Figure BDA0003060973170000063
或
Figure BDA0003060973170000064
将其计算结果与普通变性杆菌、奇异变形杆菌攻毒的小鼠血清测得值进行比较，确定普通变性杆菌、奇异变形杆菌感染血清的阴阳性临界值。
实施例2实验结果
1、膜蛋白提取与抗血清识别反应：利用细菌膜蛋白提取试剂盒从新鲜培养的奇异变形杆菌菌体中提取膜蛋白，SDS-PAGE显示提取到蛋白质，如图1所示。免疫印迹显示所提取的蛋白能与奇异变形杆菌攻毒小鼠抗血清发生反应，呈现多条条带，相对分子量在75ku至25ku，如图2所示。
2、质谱鉴定于序列分析：将对应的SDS-PAGE凝胶上的蛋白质条带切下进行LC-MS/MS分析，得到4条蛋白质序列，如SEQ ID NO.1-SEQ ID NO.4所示，将序列进行BLAST搜索，上面4条序列分别与变形杆菌的外膜孔道蛋白(Outer membrane porin，OMP)、丝氨酸水解酶(Serine hydrolase，SH)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphatedehydrogenase，GPD)、谷氨酸/天冬氨酸ABC转运体底物结合蛋白(SBP)4种蛋白具有高同源性(>82％)(表1)。
表1蛋白质信息
Figure BDA0003060973170000061
对表1所示的4种蛋白质的膜外部分进行分析(图3、图4、图5和图6)，膜外部分序列依次为：SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8。
3、基因合成及重组质粒、重组菌株构建：根据大肠杆菌密码子偏好性优化的4条蛋白质基因序列依次为：SEQ ID NO.9-SEQ ID NO.12。4条基因的5’端的“ggatcc”和3’端终止密码子taa后的“ctcgag”部分为BamHⅠ和XhoⅠ识别位点。将4条基因克隆至pET-28a以构建重组质粒，将重组质粒转化到大肠杆菌BL21(DE3)菌株以构建重组菌株。基因合成及重组质粒、重组菌株构建由生工生物工程(上海)股份有限公司完成。表达产物的预期分子量依次为40ku、43ku、39ku、34ku。
4、重组蛋白表达与反应原性分析：向上述4种BL21(DE3)重组菌株培养物加入0.1mmol/L IPTG，37℃下诱导表达3h。4个基因均有表达，如图7(M：蛋白质相对分子质量标准；1、3、5、7：诱导表达培养物裂解液沉淀；2、4、6、8：诱导表达培养物裂解液上清；1、2：rOMP；3、4：rSH；5、6：rGPD；7、8：rSBP)所示，其中rOMP与奇异变形杆菌感染的小鼠血清有强烈反应，在诱导物的上清和沉淀出现一条特异条带，从条带亮度看以包涵体表达为主，如图8(M：蛋白质相对分子质量标准；1：表达rOMP的诱导表达培养物裂解液沉淀；2：表达rOMP的诱导表达培养物裂解液上清)所示。
5、重组蛋白纯化：SDS-PAGE显示，采用Ni-NTA介质从37℃下0.1mmol/LIPTG诱导表达3h的BL21(DE3)重组菌株中纯化得到rOMP，纯度达91％，如图9(M：蛋白质相对分子质量标准；1：rOMP)所示。
6、包被抗原浓度的确定：包被抗原浓度为0.27μg/mL时，间接ELISA测得的3份阳性血清和3份阴性血清的P/N值最大。由此确定包被抗原浓度为rOMP0.27μg/mL。
表2间接ELISA抗原包被浓度
Figure BDA0003060973170000071
7、阴阳性临界值：50份阴性血清A450的X为0.558，SD为0.186，
Figure BDA0003060973170000072
Figure BDA0003060973170000073
以rOMP为包被抗原的间接ELISA方法测得的奇异变形杆菌、普通变性杆菌感染的小鼠血清的A450大于>0.930。因此，以
Figure BDA0003060973170000081
作为奇异变形杆菌、普通变性杆菌的阴阳性临界值更合适。
表4间接ELISA测得的小鼠血清的A450
Figure BDA0003060973170000082
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等均应包含在本发明的保护范围之内。
序列表
<110> 重庆市畜牧科学院
<120> 奇异变形杆菌抗原鉴定及其在检测变形杆菌感染中应用
<130> 20210506
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 371
<212> PRT
<213> Artificial Sequence
<400> 1
Met Met Lys Arg Asn Ile Leu Ala Val Val Ile Pro Ala Leu Met Phe
1 5 10 15
Ala Gly Ala Ala Asn Ala Ala Glu Met Tyr Asn Lys Asp Gly Asn Lys
20 25 30
Val Asp Ile Tyr Gly Lys Val Asp Val Arg His Tyr Phe Ala Asp Ala
35 40 45
Asp Thr Lys Gly Asp His Glu Ser Gly Asp Ala Ser Arg Ala Arg Ile
50 55 60
Gly Phe Lys Gly Glu Thr Gln Ile Asn Lys Asp Leu Thr Gly Phe Gly
65 70 75 80
Arg Phe Glu Tyr Glu Val Lys Thr Asn Thr Thr Glu Gly Glu Asn Lys
85 90 95
Ala Lys Thr Arg Leu Ala Tyr Ala Gly Leu Lys Phe Ala Asp Phe Gly
100 105 110
Ser Leu Asp Tyr Gly Arg Asn Tyr Gly Val Ile Tyr Asp Thr Asn Ala
115 120 125
Trp Thr Asp Val Leu Pro Leu Trp Gly Gly Asp Ser Met Ala Gln Thr
130 135 140
Asp Val Tyr Met Thr Ser Arg Thr Thr Gly Val Leu Thr Tyr Arg Asn
145 150 155 160
Thr Asp Met Phe Gly Tyr Val Asp Gly Leu Ser Phe Ala Leu Gln Tyr
165 170 175
Gln Gly Lys Asn Asn Glu Asn Thr Asn Asn Lys Arg Lys Asp Asn Ala
180 185 190
Glu Ala Asn Gly Asp Gly Phe Gly Phe Ser Thr Ala Tyr Asn Leu Gly
195 200 205
Trp Gly Val Thr Leu Gly Gly Gly Tyr Ser Ser Ser Ala Arg Thr Asn
210 215 220
Trp Gln Glu His Lys Ser Asp Ala Thr Gly Lys Arg Ala Glu Ala Trp
225 230 235 240
Asn Val Gly Gly Lys Phe Glu Ala Asn Asn Val Tyr Leu Ala Ala Met
245 250 255
Tyr Gly Glu Thr Arg Asn Met Asn Tyr Tyr Gly Asp Gly Ala Ile Ala
260 265 270
Asn Lys Thr Gln Asn Ile Glu Leu Thr Ala Gln Tyr Asp Phe Ala Asp
275 280 285
Leu Gly Ile Lys Pro Ser Leu Gly Tyr Val Gln Ser Lys Gly Lys Asp
290 295 300
Leu Asn Asn Gly Val Gly Gly Leu Lys Asp Asn Asn His Asp Leu Val
305 310 315 320
Lys Tyr Ile Ser Val Gly Ser Phe Tyr Lys Phe Asn Lys Asn Met Thr
325 330 335
Ala Val Val Asp Tyr Lys Ile Asn Leu Leu Asp Glu Asp Asp Phe Thr
340 345 350
Lys Ala Asn Gly Val Ala Thr Asp Asn Val Val Gly Leu Gly Leu Thr
355 360 365
Tyr Gln Phe
370
<210> 2
<211> 400
<212> PRT
<213> Artificial Sequence
<400> 2
Met Lys Thr Asn Leu Pro Ser Val Leu Ala Lys Val Thr Ala Gly Met
1 5 10 15
Gly Leu Leu Leu Ile Ile Ser Ser Pro Thr Phe Ala Val Asn Asn Pro
20 25 30
Val Pro Pro Gln Ile Glu Ala Lys Ser Tyr Val Leu Met Asp Tyr Asn
35 40 45
Ser Gly Lys Ile Leu Ala Ser Glu Lys Pro Glu Glu Arg Leu Asp Pro
50 55 60
Ala Ser Leu Thr Lys Ile Met Ser Ser Tyr Val Val Gly Gln Ala Ile
65 70 75 80
Lys Glu Gly Arg Met Ser Pro Asp Asp Ile Val Ile Val Gly Arg Asn
85 90 95
Ala Trp Ala Thr Gly Asn Pro Val Leu Lys Gly Ser Ser Leu Met Phe
100 105 110
Leu Lys Pro Gly Asp Arg Val Lys Val Ile Asp Leu Asn Arg Gly Met
115 120 125
Val Ile Gln Ser Gly Asn Asp Ala Ser Ile Ala Leu Ala Glu His Val
130 135 140
Ala Gly Ser Gln Glu Thr Phe Val Asp Leu Met Asn Thr Tyr Ala Ala
145 150 155 160
Asn Leu Gly Leu Lys Asn Thr His Phe Lys Thr Val His Gly Leu Asp
165 170 175
Ser Glu Gly Gln Tyr Thr Thr Ala Gln Asp Met Ala Leu Leu Thr Ala
180 185 190
Ala Met Ile Arg Asp Val Pro Ser Glu Tyr Asp Ile His Lys Glu Lys
195 200 205
Glu Phe Thr Phe Asn Asn Ile Arg Gln Pro Asn Arg Asn Arg Leu Leu
210 215 220
Trp Asn Lys Asn Met Asn Val Asp Gly Val Lys Thr Gly His Thr Asn
225 230 235 240
Gly Ala Gly Tyr Asn Leu Val Ser Ser Ala Thr Glu Gly Asn Met Arg
245 250 255
Leu Ile Ala Val Val Leu Gly Ala Pro Ser Asp Lys Val Arg Phe Ala
260 265 270
Glu Ser Glu Arg Leu Leu Gly Trp Gly Phe Arg Phe Phe Glu Thr Val
275 280 285
Thr Pro Val Lys Glu Asn Val Pro Leu Thr Thr Gln Lys Val Trp Tyr
290 295 300
Gly Asp Lys Gly Glu Val Ala Leu Gly Val Ala Lys Asp Ala Ser Ile
305 310 315 320
Thr Ile Pro Lys Gly Glu Leu Lys Asn Leu Lys Ala Ser Phe Thr Leu
325 330 335
Thr Asn Pro Glu Leu Glu Ala Pro Leu Thr Gln Asn Gln Val Val Gly
340 345 350
Thr Val Asn Phe Leu Leu Asn Asp Glu Val Ile Glu Gln Arg Pro Leu
355 360 365
Val Val Lys Glu Ala Val Glu Glu Gly Gly Phe Phe Ser Arg Ile Trp
370 375 380
Asp Phe Val Val Lys Thr Val Ser Gly Trp Phe Asn Ala Leu Phe Gly
385 390 395 400
<210> 3
<211> 331
<212> PRT
<213> Artificial Sequence
<400> 3
Met Thr Ile Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Ile
1 5 10 15
Val Phe Arg Ala Ala Gln Glu Arg Ser Asp Ile Glu Ile Val Gly Ile
20 25 30
Asn Asp Leu Leu Asp Ala Glu Tyr Met Ala Tyr Met Leu Lys Tyr Asp
35 40 45
Ser Thr His Gly Arg Phe Asn Gly Thr Val Glu Val Lys Asp Gly His
50 55 60
Leu Val Val Asn Gly Lys Thr Ile Arg Val Thr Ser Glu Arg Asp Pro
65 70 75 80
Ala Asn Leu Lys Trp Asn Glu Ile Gly Val Asp Val Val Ala Glu Ala
85 90 95
Thr Gly Leu Phe Leu Thr Asp Glu Thr Ala Arg Lys His Ile Gln Ala
100 105 110
Gly Ala Lys Lys Val Val Leu Thr Gly Pro Ser Lys Asp Ser Thr Pro
115 120 125
Met Phe Val Met Gly Val Asn His Lys Ser Tyr Ala Gly Gln Asp Ile
130 135 140
Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys
145 150 155 160
Val Ile Asn Asp Lys Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val
165 170 175
His Ala Thr Thr Ala Thr Gln Arg Thr Val Asp Gly Pro Ser Met Lys
180 185 190
Asp Trp Arg Gly Gly Arg Gly Ala Ser Gln Asn Ile Ile Pro Ser Ser
195 200 205
Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly
210 215 220
Lys Leu Thr Gly Met Ser Phe Arg Val Pro Thr Pro Asn Val Ser Val
225 230 235 240
Val Asp Leu Thr Ala Arg Leu Glu Lys Pro Ala Thr Tyr Ala Gln Ile
245 250 255
Cys Glu Ala Ile Lys Glu Ala Ala Glu Gly Glu Leu Lys Gly Val Leu
260 265 270
Gly Tyr Thr Glu Asp Ala Val Val Ser Thr Asp Phe Asn Gly Glu Val
275 280 285
Leu Thr Ser Val Phe Asp Ala Lys Ala Gly Ile Ala Leu Asn Asp Asn
290 295 300
Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Val Gly Tyr Ser Asn
305 310 315 320
Lys Val Leu Asp Leu Ile Ser His Ile Ser Lys
325 330
<210> 4
<211> 297
<212> PRT
<213> Artificial Sequence
<400> 4
Met Arg Met Arg Lys Leu Ala Leu Ser Leu Met Ile Leu Gly Ala Ala
1 5 10 15
Gly Ala Val Gln Ala Glu Glu Leu Thr Gly Thr Leu Lys Lys Ile Asn
20 25 30
Asp Asn Asp Ile Ile Ile Val Gly His Arg Glu Ser Ser Val Pro Phe
35 40 45
Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr
50 55 60
Ser Asn Leu Ile Val Glu Ala Val Lys Glu Lys Leu Gly Lys Pro Asn
65 70 75 80
Leu Gln Val Arg Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu
85 90 95
Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn
100 105 110
Leu Ala Arg Gln Gln Gln Ala Asp Phe Ser Asn Thr Ile Phe Ile Val
115 120 125
Gly Thr Arg Leu Leu Ala Lys Lys Asp Ser Gly Ile Lys Asp Phe Ala
130 135 140
Asp Leu Ala Gly Lys Asn Val Val Val Thr Ser Gly Thr Thr Ser Glu
145 150 155 160
Met Leu Leu Asn Lys Leu Asn Asp Glu Lys Lys Met Asn Met Arg Ile
165 170 175
Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly
180 185 190
Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg
195 200 205
Ala Lys Ala Lys Lys Pro Ala Leu Trp Glu Ile Val Gly Thr Pro Gln
210 215 220
Ser Glu Glu Ala Tyr Gly Cys Met Leu Arg Lys Gly Asp Thr Gln Phe
225 230 235 240
Lys Ser Leu Ile Asp Glu Thr Ile Ala Asn Ala Gln Lys Ser Gly Ala
245 250 255
Ala Glu Lys Ser Phe Asn Arg Trp Phe Asn Ala Pro Ile Pro Pro Lys
260 265 270
Asn Leu Asn Leu Glu Phe Thr Ile Ser Asp Glu Met Lys Ala Leu Phe
275 280 285
Ala Ser Pro Asn Asp Lys Ala Leu Asn
290 295
<210> 5
<211> 335
<212> PRT
<213> Artificial Sequence
<400> 5
Gly Lys Val Asp Val Arg His Tyr Phe Ala Asp Ala Asp Thr Lys Gly
1 5 10 15
Asp His Glu Ser Gly Asp Ala Ser Arg Ala Arg Ile Gly Phe Lys Gly
20 25 30
Glu Thr Gln Ile Asn Lys Asp Leu Thr Gly Phe Gly Arg Phe Glu Tyr
35 40 45
Glu Val Lys Thr Asn Thr Thr Glu Gly Glu Asn Lys Ala Lys Thr Arg
50 55 60
Leu Ala Tyr Ala Gly Leu Lys Phe Ala Asp Phe Gly Ser Leu Asp Tyr
65 70 75 80
Gly Arg Asn Tyr Gly Val Ile Tyr Asp Thr Asn Ala Trp Thr Asp Val
85 90 95
Leu Pro Leu Trp Gly Gly Asp Ser Met Ala Gln Thr Asp Val Tyr Met
100 105 110
Thr Ser Arg Thr Thr Gly Val Leu Thr Tyr Arg Asn Thr Asp Met Phe
115 120 125
Gly Tyr Val Asp Gly Leu Ser Phe Ala Leu Gln Tyr Gln Gly Lys Asn
130 135 140
Asn Glu Asn Thr Asn Asn Lys Arg Lys Asp Asn Ala Glu Ala Asn Gly
145 150 155 160
Asp Gly Phe Gly Phe Ser Thr Ala Tyr Asn Leu Gly Trp Gly Val Thr
165 170 175
Leu Gly Gly Gly Tyr Ser Ser Ser Ala Arg Thr Asn Trp Gln Glu His
180 185 190
Lys Ser Asp Ala Thr Gly Lys Arg Ala Glu Ala Trp Asn Val Gly Gly
195 200 205
Lys Phe Glu Ala Asn Asn Val Tyr Leu Ala Ala Met Tyr Gly Glu Thr
210 215 220
Arg Asn Met Asn Tyr Tyr Gly Asp Gly Ala Ile Ala Asn Lys Thr Gln
225 230 235 240
Asn Ile Glu Leu Thr Ala Gln Tyr Asp Phe Ala Asp Leu Gly Ile Lys
245 250 255
Pro Ser Leu Gly Tyr Val Gln Ser Lys Gly Lys Asp Leu Asn Asn Gly
260 265 270
Val Gly Gly Leu Lys Asp Asn Asn His Asp Leu Val Lys Tyr Ile Ser
275 280 285
Val Gly Ser Phe Tyr Lys Phe Asn Lys Asn Met Thr Ala Val Val Asp
290 295 300
Tyr Lys Ile Asn Leu Leu Asp Glu Asp Asp Phe Thr Lys Ala Asn Gly
305 310 315 320
Val Ala Thr Asp Asn Val Val Gly Leu Gly Leu Thr Tyr Gln Phe
325 330 335
<210> 6
<211> 362
<212> PRT
<213> Artificial Sequence
<400> 6
Ala Lys Ser Tyr Val Leu Met Asp Tyr Asn Ser Gly Lys Ile Leu Ala
1 5 10 15
Ser Glu Lys Pro Glu Glu Arg Leu Asp Pro Ala Ser Leu Thr Lys Ile
20 25 30
Met Ser Ser Tyr Val Val Gly Gln Ala Ile Lys Glu Gly Arg Met Ser
35 40 45
Pro Asp Asp Ile Val Ile Val Gly Arg Asn Ala Trp Ala Thr Gly Asn
50 55 60
Pro Val Leu Lys Gly Ser Ser Leu Met Phe Leu Lys Pro Gly Asp Arg
65 70 75 80
Val Lys Val Ile Asp Leu Asn Arg Gly Met Val Ile Gln Ser Gly Asn
85 90 95
Asp Ala Ser Ile Ala Leu Ala Glu His Val Ala Gly Ser Gln Glu Thr
100 105 110
Phe Val Asp Leu Met Asn Thr Tyr Ala Ala Asn Leu Gly Leu Lys Asn
115 120 125
Thr His Phe Lys Thr Val His Gly Leu Asp Ser Glu Gly Gln Tyr Thr
130 135 140
Thr Ala Gln Asp Met Ala Leu Leu Thr Ala Ala Met Ile Arg Asp Val
145 150 155 160
Pro Ser Glu Tyr Asp Ile His Lys Glu Lys Glu Phe Thr Phe Asn Asn
165 170 175
Ile Arg Gln Pro Asn Arg Asn Arg Leu Leu Trp Asn Lys Asn Met Asn
180 185 190
Val Asp Gly Val Lys Thr Gly His Thr Asn Gly Ala Gly Tyr Asn Leu
195 200 205
Val Ser Ser Ala Thr Glu Gly Asn Met Arg Leu Ile Ala Val Val Leu
210 215 220
Gly Ala Pro Ser Asp Lys Val Arg Phe Ala Glu Ser Glu Arg Leu Leu
225 230 235 240
Gly Trp Gly Phe Arg Phe Phe Glu Thr Val Thr Pro Val Lys Glu Asn
245 250 255
Val Pro Leu Thr Thr Gln Lys Val Trp Tyr Gly Asp Lys Gly Glu Val
260 265 270
Ala Leu Gly Val Ala Lys Asp Ala Ser Ile Thr Ile Pro Lys Gly Glu
275 280 285
Leu Lys Asn Leu Lys Ala Ser Phe Thr Leu Thr Asn Pro Glu Leu Glu
290 295 300
Ala Pro Leu Thr Gln Asn Gln Val Val Gly Thr Val Asn Phe Leu Leu
305 310 315 320
Asn Asp Glu Val Ile Glu Gln Arg Pro Leu Val Val Lys Glu Ala Val
325 330 335
Glu Glu Gly Gly Phe Phe Ser Arg Ile Trp Asp Phe Val Val Lys Thr
340 345 350
Val Ser Gly Trp Phe Asn Ala Leu Phe Gly
355 360
<210> 7
<211> 331
<212> PRT
<213> Artificial Sequence
<400> 7
Met Thr Ile Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Ile
1 5 10 15
Val Phe Arg Ala Ala Gln Glu Arg Ser Asp Ile Glu Ile Val Gly Ile
20 25 30
Asn Asp Leu Leu Asp Ala Glu Tyr Met Ala Tyr Met Leu Lys Tyr Asp
35 40 45
Ser Thr His Gly Arg Phe Asn Gly Thr Val Glu Val Lys Asp Gly His
50 55 60
Leu Val Val Asn Gly Lys Thr Ile Arg Val Thr Ser Glu Arg Asp Pro
65 70 75 80
Ala Asn Leu Lys Trp Asn Glu Ile Gly Val Asp Val Val Ala Glu Ala
85 90 95
Thr Gly Leu Phe Leu Thr Asp Glu Thr Ala Arg Lys His Ile Gln Ala
100 105 110
Gly Ala Lys Lys Val Val Leu Thr Gly Pro Ser Lys Asp Ser Thr Pro
115 120 125
Met Phe Val Met Gly Val Asn His Lys Ser Tyr Ala Gly Gln Asp Ile
130 135 140
Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys
145 150 155 160
Val Ile Asn Asp Lys Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val
165 170 175
His Ala Thr Thr Ala Thr Gln Arg Thr Val Asp Gly Pro Ser Met Lys
180 185 190
Asp Trp Arg Gly Gly Arg Gly Ala Ser Gln Asn Ile Ile Pro Ser Ser
195 200 205
Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly
210 215 220
Lys Leu Thr Gly Met Ser Phe Arg Val Pro Thr Pro Asn Val Ser Val
225 230 235 240
Val Asp Leu Thr Ala Arg Leu Glu Lys Pro Ala Thr Tyr Ala Gln Ile
245 250 255
Cys Glu Ala Ile Lys Glu Ala Ala Glu Gly Glu Leu Lys Gly Val Leu
260 265 270
Gly Tyr Thr Glu Asp Ala Val Val Ser Thr Asp Phe Asn Gly Glu Val
275 280 285
Leu Thr Ser Val Phe Asp Ala Lys Ala Gly Ile Ala Leu Asn Asp Asn
290 295 300
Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Val Gly Tyr Ser Asn
305 310 315 320
Lys Val Leu Asp Leu Ile Ser His Ile Ser Lys
325 330
<210> 8
<211> 276
<212> PRT
<213> Artificial Sequence
<400> 8
Glu Glu Leu Thr Gly Thr Leu Lys Lys Ile Asn Asp Asn Asp Ile Ile
1 5 10 15
Ile Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn
20 25 30
Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn Leu Ile Val
35 40 45
Glu Ala Val Lys Glu Lys Leu Gly Lys Pro Asn Leu Gln Val Arg Leu
50 55 60
Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr
65 70 75 80
Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Leu Ala Arg Gln Gln
85 90 95
Gln Ala Asp Phe Ser Asn Thr Ile Phe Ile Val Gly Thr Arg Leu Leu
100 105 110
Ala Lys Lys Asp Ser Gly Ile Lys Asp Phe Ala Asp Leu Ala Gly Lys
115 120 125
Asn Val Val Val Thr Ser Gly Thr Thr Ser Glu Met Leu Leu Asn Lys
130 135 140
Leu Asn Asp Glu Lys Lys Met Asn Met Arg Ile Ile Ser Ala Lys Asp
145 150 155 160
His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala Val Ala Phe
165 170 175
Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys
180 185 190
Pro Ala Leu Trp Glu Ile Val Gly Thr Pro Gln Ser Glu Glu Ala Tyr
195 200 205
Gly Cys Met Leu Arg Lys Gly Asp Thr Gln Phe Lys Ser Leu Ile Asp
210 215 220
Glu Thr Ile Ala Asn Ala Gln Lys Ser Gly Ala Ala Glu Lys Ser Phe
225 230 235 240
Asn Arg Trp Phe Asn Ala Pro Ile Pro Pro Lys Asn Leu Asn Leu Glu
245 250 255
Phe Thr Ile Ser Asp Glu Met Lys Ala Leu Phe Ala Ser Pro Asn Asp
260 265 270
Lys Ala Leu Asn
275
<210> 9
<211> 1020
<212> DNA
<213> Artificial Sequence
<400> 9
ggatccggta aagttgatgt tcgtcactac ttcgcggatg cggataccaa aggtgatcac 60
gaaagcggtg atgcgagccg tgcgcgtatc ggtttcaaag gtgaaaccca gatcaacaaa 120
gatctgaccg gcttcggccg tttcgaatat gaagttaaaa ccaacaccac cgaaggtgaa 180
aacaaagcga aaacccgtct ggcgtacgca ggcctgaaat tcgcggattt cggttctctg 240
gattacggtc gtaactacgg tgttatctac gataccaacg cgtggaccga tgttctgccg 300
ctgtggggcg gcgatagcat ggctcagacc gatgtttaca tgacctctcg taccaccggc 360
gttctgacct accgtaacac cgatatgttc ggttacgtgg atggtctgag cttcgcgctg 420
cagtaccagg gtaaaaacaa cgaaaacacc aacaacaaac gtaaagataa cgcggaagcg 480
aacggtgatg gtttcggttt cagcaccgcg tacaacctgg gttggggtgt taccctgggt 540
ggcggttact ctagcagcgc gcgtaccaac tggcaggaac acaaatctga tgcgaccggc 600
aaacgtgcgg aagcgtggaa cgttggtggt aaattcgaag cgaacaacgt gtacctggcg 660
gcgatgtacg gtgaaacccg taacatgaac tactacggcg atggcgcgat cgcgaacaaa 720
acccagaaca tcgaactgac cgcgcagtac gatttcgcgg atctgggtat caaaccgagc 780
ctgggttacg ttcagtctaa aggtaaagat ctgaacaacg gtgttggtgg cctgaaagat 840
aacaaccacg atctggttaa atacatctct gttggtagct tctacaaatt caacaaaaac 900
atgaccgcgg ttgttgatta caaaattaac ctgctggatg aagatgattt caccaaagcg 960
aacggtgttg cgaccgacaa cgttgttggt ctgggtctga cctaccagtt ctaactcgag 1020
<210> 10
<211> 1101
<212> DNA
<213> Artificial Sequence
<400> 10
ggatccgcta aaagctacgt tctgatggat tacaactctg gtaaaatcct ggcttctgaa 60
aaaccggaag aacgtctgga cccggcctct ctgaccaaaa tcatgagctc ttacgttgtt 120
ggtcaggcga tcaaagaagg ccgtatgagc ccggatgata tcgttattgt tggtcgtaac 180
gcttgggcga ccggtaaccc ggttctgaaa ggttcttctc tgatgttcct gaaaccgggt 240
gatcgtgtta aagttatcga tctgaaccgc ggcatggtta tccagagcgg caacgatgcg 300
agcatcgctc tggcggaaca cgttgcgggt agccaggaaa ccttcgttga tctgatgaac 360
acctacgcgg caaacctggg tctgaaaaac acccacttca aaaccgttca cggcctggat 420
tctgaaggcc agtacaccac cgcgcaggat atggcgctgc tgaccgcggc aatgatccgt 480
gatgttccga gcgaatacga tatccacaaa gaaaaagaat ttaccttcaa caacatccgt 540
cagccgaacc gtaaccgcct gctgtggaac aaaaacatga acgttgacgg cgttaaaacc 600
ggtcacacca acggcgcggg ctacaacctg gttagctctg cgactgaagg taacatgcgt 660
ctgatcgcgg tggttctggg tgcgccgtct gataaagttc gtttcgcgga atctgaacgc 720
ctgctgggtt ggggtttccg tttcttcgaa accgttaccc cggttaaaga aaacgttccg 780
ctgaccaccc agaaagtttg gtacggcgat aaaggcgaag ttgcgctggg cgttgcaaaa 840
gatgcgtcta ttaccattcc gaaaggtgaa ctgaaaaacc tgaaagctag cttcaccctg 900
accaacccgg aactggaagc accgctgacc cagaaccagg ttgttggcac cgtgaacttc 960
ctgctgaacg acgaagttat cgaacagcgc ccgctggttg ttaaagaagc ggttgaagaa 1020
ggtggcttct tctctcgtat ctgggatttc gttgttaaaa ccgttagcgg ctggttcaac 1080
gcgctgttcg gctaactcga g 1101
<210> 11
<211> 1008
<212> DNA
<213> Artificial Sequence
<400> 11
ggatccatga ccatcaaagt tggtatcaac ggcttcggtc gtatcggtcg tatcgttttc 60
cgtgcggcgc aggaacgtag cgatatcgaa atcgttggta tcaacgatct gctggatgcg 120
gaatacatgg cgtacatgct gaaatacgat agcacccacg gccgtttcaa cggtaccgtt 180
gaagttaaag atggccacct ggttgttaac ggtaaaacca tccgcgttac ctctgaacgt 240
gatccggcga acctgaaatg gaacgaaatt ggtgttgatg ttgttgcgga agcgaccggc 300
ctgttcctga ccgatgaaac cgcgcgtaaa cacatccagg cgggcgcgaa aaaagttgtt 360
ctgaccggtc cgagcaaaga tagcaccccg atgttcgtta tgggtgttaa ccacaaatct 420
tacgcgggtc aggatatcgt tagcaacgcg agctgcacca ccaactgcct ggcgccgctg 480
gcgaaagtta tcaacgataa attcggtatc gttgaaggcc tgatgaccac cgttcacgct 540
accaccgcga cccagcgtac cgttgatggc ccgtctatga aagattggcg tggtggtcgt 600
ggcgcgtccc agaacatcat cccgagcagc accggcgcgg cgaaagcggt tggtaaagtt 660
atcccggaac tgaacggtaa actgaccggt atgtctttcc gtgttccgac cccgaacgtt 720
agcgttgttg atctgaccgc gcgtctggaa aaaccggcga cctacgcgca gatctgcgaa 780
gcgatcaaag aagcggcgga aggtgaactg aaaggtgttc tgggttacac cgaagatgcg 840
gttgttagca ccgatttcaa cggtgaagtt ctgacctctg ttttcgatgc aaaagcgggc 900
atcgcgctga acgataactt cgttaaactg gttagctggt acgataacga agtgggctac 960
agcaacaaag ttctggatct gatcagccac atctctaaat aactcgag 1008
<210> 12
<211> 843
<212> DNA
<213> Artificial Sequence
<400> 12
ggatccgaag aactcactgg taccttaaaa aaaatcaacg ataacgatat tattattgtt 60
ggtcatcgtg aatcctctgt ccctttttca tactacgata atcaacaaaa agtggtcgga 120
tactctcaag actattccaa cctgattgtt gaggcagtaa aagaaaaatt aggcaaacct 180
aacctacaag ttcgccttat ccctatcact tctcagaatc gaattccact attacaaaat 240
ggtacatttg actttgagtg tggctcaacg accaataatc tcgctcgcca acaacaagct 300
gatttctcga atactatttt tattgttggt acacgtttac ttgcgaaaaa agacagtgga 360
attaaagatt ttgctgatct tgcgggcaaa aatgtggttg tcacgtcagg caccacatct 420
gaaatgttac ttaacaagct caatgatgag aaaaaaatga atatgcgcat tattagcgca 480
aaagatcatg gcgactcatt ccgtacactt gaatcagggc gtgccgttgc ttttatgatg 540
gatgacgcct tattagcggg tgaacgtgct aaagcgaaaa aacctgcgtt atgggagatt 600
gtgggaacac ctcaatcgga agaagcttat ggctgtatgt tacgcaaagg tgatacacaa 660
tttaagtcat taattgatga aaccattgct aatgcgcaaa aatctggcgc agcagaaaaa 720
tcgttcaacc gttggtttaa tgccccaatt ccaccgaaaa acctcaattt agaattcact 780
atctcggatg aaatgaaagc actctttgca tcaccaaatg ataaagcatt gaactaactc 840
gag 843

Claims (10)

1.一种奇异变形杆菌膜蛋白的膜外部分蛋白，其特征在于，所述膜外部分蛋白的氨基酸序列依次由SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8组成。
2.一种包括优化后的权利要求1所述膜外部分蛋白的重组质粒，其特征在于，所述重组质粒的核苷酸序列依次由SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12组成。
3.根据权利要求2所述的重组质粒，其特征在于，所述重组质粒的制备方法是依次将所述SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12的核苷酸序列克隆至pETET-28a中。
4.一种利用权利要求2或3所述的重组质粒制备得到的基因工程菌株。
5.权利要求4所述的基因工程菌株产生的重组蛋白。
6.权利要求5所述的重组蛋白在制备检测奇异变形杆菌或普通变形杆菌的试剂盒中的应用。
7.利用权利要求5所述的重组蛋白检测奇异变形杆菌或普通变形杆菌的方法，其特征在于，检测样本为奇异变形杆菌和/或普通变形杆菌和/或伪结核棒状杆菌和/或金黄色葡萄球菌和/或大肠杆菌和/或化脓隐秘杆菌，具体包括如下步骤：
(1)纯化所述重组蛋白；
(2)以rOMP包被所述纯化重组蛋白；
(3)利用间接ELISA方法检测血清的A450；
(4)当所述
Figure FDA0003060973160000011
时，检测为奇异变形杆菌或普通变形杆菌。
8.根据权利要求7所述的方法，其特征在于，步骤(2)包被过程中加入HRP标记的山羊抗小鼠IgG抗体。
9.根据权利要求7所述的方法，其特征在于，所述
Figure FDA0003060973160000012
10.利用权利要求5所述的重组蛋白将奇异变形杆菌、普通变形杆菌与伪结核棒状杆菌、金黄色葡萄球菌、大肠杆菌、化脓隐秘杆菌区分开来的方法，其特征在于，利用所述重组蛋白作为抗原，所述抗原作为捕获剂被固相载体固定，用于捕获特异性抗体，所述特异性抗体为HRP标记的山羊抗小鼠IgG抗体；通过间接ELISA方法检测所述山羊抗小鼠IgG抗体，测定A450区分出奇异变形杆菌和普通变形杆菌。
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