CN113321739B - 一种covid-19亚单位疫苗及其制备方法与应用 - Google Patents

一种covid-19亚单位疫苗及其制备方法与应用 Download PDF

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CN113321739B
CN113321739B CN202110421895.6A CN202110421895A CN113321739B CN 113321739 B CN113321739 B CN 113321739B CN 202110421895 A CN202110421895 A CN 202110421895A CN 113321739 B CN113321739 B CN 113321739B
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李展如
刘洪恩
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Guangdong Keguanda Pharmaceutical Technology Co ltd
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Abstract

本发明通过基因工程手段将新型冠状病毒(SARS‑CoV‑2)刺突蛋白受体结合结构域(RBD)与人免疫球蛋白hFc域或His标签结合得到的融合蛋白RBD‑hFc/RBD‑His便于提取与纯化，质量稳定可控，耗时短且可大规模生产。利用本发明的融合蛋白与佐剂配置得到的疫苗组合物，可以增加疫苗的溶解度、稳定性，增强疫苗免疫原性，延长疫苗在体内半衰期。本发明的融合蛋白及其疫苗组合物可抑制SARS‑CoV‑2野生型和/或变异型毒株的复制、传播或阻止其在宿主体内定居，从而能有效预防和/或治疗SARS‑CoV‑2野生型和/或变异型毒株引起的新型冠状病毒肺炎。

Description

一种COVID-19亚单位疫苗及其制备方法与应用
技术领域
本发明涉及生物技术领域，尤其涉及一种COVID-19亚单位疫苗及其制备方法与应用。
背景技术
SARS-CoV-2是一种正义单链RNA病毒，直径约80～120nm，与SARS-CoV和MERS-CoV同属于β冠状病毒属。其基因组长度约为29.8Kb，共编码四种结构蛋白：刺突蛋白(Spike，S)、小囊膜蛋白(Envelope，E)、膜蛋白(Membrane，M)和核衣壳蛋白(Nucleocapsid，N)(Immunity,2020.53(6):p.1315-1330)。其中S蛋白位于病毒表面，由N-末端S1结构域和C-末端S2结构域组成。位于S1亚基中的受体结合结构域(receptor-binding domain，RBD)能与细胞表面的血管紧张素转化酶2(angiotensin-converting enzyme 2，ACE2)结合，在介导病毒进入细胞的过程中发挥重要作用。研究表明SARS-CoV-2RBD与ACE2的结合能力比SARS-CoV高约10～20倍(Science,2020.367(6483):p.1260-1263)，此提示RBD作为开发亚单位疫苗抗原的可行性。目前基于RBD蛋白设计的疫苗种类主要包括重组蛋白疫苗(Vaccine,2020.38(46):p.7205-7212)、DNA疫苗(Science,2020.369(6505):p.806-811)和mRNA疫苗(Cell,2020.182(5):p.1271-1283)等。
一般而言，进行蛋白疫苗抗原设计时，SARS-CoV-2RBD的抗原长度可能会影响疫苗诱导的免疫反应，因此需要研究不同长度的RBD以筛选最优的序列。此外，RBD分子量小(～21KDa)，具有免疫原性差、诱导产生的免疫反应持续时间较短、需要多次接种等缺点，不适合单独制备成疫苗，且体外获得的RBD蛋白不易提取与纯化，因此，如何得到亚单位疫苗并提高亚单位疫苗的有效性与安全性是急需解决的技术问题。利用基因工程技术将SARS-CoV-2S蛋白的抗原，通过连接子(linker)与人免疫球蛋白Fc(hFc)域或组氨酸(His)标签连接，制得抗SARS-CoV-2的融合蛋白疫苗，在稳定性、溶解度和体内半衰期等方面都能得到一定程度的改善，并易于规模生产和提取分离。融合蛋白中连接子的有无及种类会对蛋白质空间结构、溶解性、稳定性、蛋白质表面上氨基酸暴露数量、序列柔韧性及抗原表位等方面造成影响，甚至可能会增强蛋白的某些生物学活性(J Med Virol,2020)，因此，RBD重组蛋白疫苗连接子的有无及连接子的种类对RBD抗原在体内免疫原性可能有较大的影响。
从结构上来看，SARS-CoV-2毒株具有Furin蛋白酶切割位点，经Furin切割后暴露出开放结构域可极大提高S蛋白与ACE2的结合能力，因此Furin位点附近的突变可能会对毒株的结合能力及传染性造成影响，其次是RBD的6个关键交界面氨基酸与ACE2结合，也可能影响毒株的结合能力及传染性。2020年12月公布的B.1.1.7变异型毒株产生了RBD交界面氨基酸的N501Y与Furin蛋白酶切割位点附近A570D的关键突变，这对病毒的传播速度可能具有影响(Cell.2021；doi:10.1016/j.cell.2021.02.042)；501Y.V2变异型毒株产生的E484K不仅使其传染能力增强，还可能逃脱中和抗体(Nature.2021；doi:10.1038/s41586-021-03471-w)。针对SARS-CoV-2野生型毒性的RBD制备的疫苗对变异型毒株是否具有预防或治疗作用仍待进一步研究，但不能排除作用减弱甚至无效，因此，急需设计一种能同时针对野生型与变异型SARS-CoV-2的联合疫苗。
发明内容
本发明的目的在于克服现有技术的不足，提供一种COVID-19亚单位疫苗及其制备方法与应用，可以有效地克服野生型SARS-CoV-2和/或变异型SARS-CoV-2导致的疫情暴发及其对防治COVID-19带来的巨大困难。
为实现上述目的，本发明采取的技术方案为：提供一种融合蛋白，所述融合蛋白为将新型冠状病毒刺突蛋白的受体结合结构域RBD通过连接子和人免疫球蛋白hFc域或His标签多肽片段连接得到。
本发明通过基因工程手段将蛋白RBD与人免疫球蛋白hFc域或His标签结合得到的融合蛋白RBD-hFc/RBD-His便于提取与纯化，质量稳定可控，耗时短且可大规模生产。利用融合蛋白RBD-hFc/RBD-His与佐剂配制得到的疫苗组合物，可以增加疫苗的溶解度、稳定性，增强疫苗免疫原性，延长疫苗在体内半衰期。本发明的融合蛋白及其疫苗组合物可抑制SARS-CoV-2野生型和/或变异型毒株的复制、传播或阻止其在宿主体内定居，从而能有效预防和/或治疗野生型和/或变异型毒株引起的新型冠状病毒肺炎。
作为本发明所述融合蛋白的优选实施方式，所述连接子的氨基酸序列为ADDDDK、AAA或A。
作为本发明所述融合蛋白的优选实施方式，所述新型冠状病毒包括野生型毒株、B.1.1.7变异型毒株和501Y.V2变异型毒株；
所述野生型毒株的GeneID为NC_045512；
所述B.1.1.7变异型毒株的GeneID为MW735442，所述B.1.1.7变异型毒株产生了RBD交界面氨基酸的N501Y与Furin蛋白酶切割位点附近A570D的突变；
所述501Y.V2变异型毒株的GeneID为MW789246，所述501Y.V2变异型毒株产生了K417N、E484K和N501Y突变。
作为本发明所述融合蛋白的优选实施方式，所述RBD的氨基酸序列为：新型冠状病毒刺突蛋白氨基酸序列的第319-593位或新型冠状病毒刺突蛋白氨基酸序列的第319-593位中的任意一段；
所述新型冠状病毒刺突蛋白氨基酸序列的第319-593位的氨基酸序列如SEQ IDNo.1所示。
作为本发明所述融合蛋白的优选实施方式，所述RBD的氨基酸序列为：新型冠状病毒刺突蛋白氨基酸序列的第319-541位、第331-593位或第319-583位。
作为本发明所述融合蛋白的优选实施方式，所述人免疫球蛋白hFc域包括来自人源IgG、IgA、IgD、IgE和IgM的Fc片段中的至少一种。
作为本发明所述融合蛋白的优选实施方式，所述人免疫球蛋白hFc域的氨基酸序列如SEQ ID No.33所示，所述人免疫球蛋白hFc域的核苷酸序列(HD-13)如SEQ ID No.32所示。
作为本发明所述融合蛋白的优选实施方式，所述His标签多肽片段含有4～14个组氨酸残基，优选地，含有10个组氨酸残基。
作为本发明所述融合蛋白的优选实施方式，所述融合蛋白的氨基酸序列如SEQ IDNo.3、SEQ ID No.5、SEQ ID No.7、SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ IDNo.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.25、SEQ IDNo.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.35和SEQ ID No.37任一所述。
作为本发明所述融合蛋白的优选实施方式，所述融合蛋白包括17种：HD-R1、HD-R2、HD-05、HD-R3、HD-01、HD-02、HD-03、HD-04、HD-06、HD-07、HD-08、HD-09、HD-10、HD-11、HD-12、HD-S1和HD-S，所述融合蛋白的核苷酸序列和氨基酸序列如表1所示：
表1：17种融合蛋白的核苷酸序列和氨基酸序列
融合蛋白编号 核苷酸序列 氨基酸序列
HD-R1 SEQ ID No.2 SEQ ID No.3
HD-R2 SEQ ID No.4 SEQ ID No.5
HD-05 SEQ ID No.6 SEQ ID No.7
HD-R3 SEQ ID No.8 SEQ ID No.9
HD-01 SEQ ID No.10 SEQ ID No.11
HD-02 SEQ ID No.12 SEQ ID No.13
HD-03 SEQ ID No.14 SEQ ID No.15
HD-04 SEQ ID No.16 SEQ ID No.17
HD-06 SEQ ID No.18 SEQ ID No.19
HD-07 SEQ ID No.20 SEQ ID No.21
HD-08 SEQ ID No.22 SEQ ID No.23
HD-09 SEQ ID No.24 SEQ ID No.25
HD-10 SEQ ID No.26 SEQ ID No.27
HD-11 SEQ ID No.28 SEQ ID No.29
HD-12 SEQ ID No.30 SEQ ID No.31
HD-S1 SEQ ID No.34 SEQ ID No.35
HD-S SEQ ID No.36 SEQ ID No.37
本发明同时提供了编码所述融合蛋白的核酸分子。
作为本发明所述核酸分子的优选实施方式，所述核酸分子的序列是指SEQ IDNo.2、SEQ ID No.4、SEQ ID No.6、SEQ ID No.8、SEQ ID No.10、SEQ ID No.12、SEQ IDNo.14、SEQ ID No.16、SEQ ID No.18、SEQ ID No.20、SEQ ID No.22、SEQ ID No.24、SEQ IDNo.26、SEQ ID No.28、SEQ ID No.30、SEQ ID No.34和SEQ ID No.36任一所示的多核苷酸序列或其简并序列。
本发明同时提供包含所述核酸分子的载体。
作为本发明所述载体的优选实施方式，所述载体是将所述核酸分子连接到质粒pcDNA3.1中得到。
本发明同时提供包含所述核酸分子或所述载体的细胞。
作为本发明所述细胞的优选实施方式，所述细胞为将核酸分子或载体转入宿主细胞Expi293F中得到。
本发明同时提供一种疫苗组合物，所述疫苗组合物包含所述融合蛋白、所述核酸分子或所述的载体。
本发明同时提供一种多价疫苗组合物，所述多价疫苗组合物包含至少两种所述融合蛋白。
本发明同时提供了一种针对野生型与变异型SARS-CoV-2不同氨基酸序列的RBD融合蛋白疫苗及其联合疫苗(包括但不限制于二价、三价疫苗)，这些疫苗对野生型毒株和/或变异型毒株同时具有免疫反应作用。这些融合蛋白疫苗可通过重组蛋白技术、载体表达技术、核酸技术实现，并进行组合作为联合疫苗、多价疫苗应用。
为考察单一疫苗免疫时对变异型毒株的抵抗作用，及三种疫苗联合免疫时对野生型毒株和变异型毒株的免疫反应作用，针对野生型毒株设计的疫苗有9种：HD-R1、HD-R2、HD-R3、HD-04、HD-05、HD-07、HD-10、HD-S1和HD-S；针对变异型毒株501Y.V2(K417N、E484K和N501Y)设计的疫苗有4种：HD-01、HD-06、HD-09和HD-12；针对变异型毒株B.1.1.7(A570D和N501Y)设计的疫苗有4种：HD-02、HD-03、HD-08和HD-11。以上的17种蛋白疫苗可以单独或任意两种或者三种通过不同比例混合，制备成疫苗。优选含有针对野生型毒株、针对变异型毒株501Y.V2和针对变异型毒株B.1.1.7三类疫苗中的两种或者三种相互混合；融合蛋白质量比包括1:1:1、0:1:1、1:0:1、1:1:0、1:0.5:1、0.5:1:1、1:1:0.5、2:1:1、1:2:1与1:1:2，优选的，所述融合蛋白质量比为1:1:1、0:1:1、1:0:1或1:1:0。
重组蛋白疫苗技术是基于已发布的病毒基因序列，将病毒目的抗原基因通过重组的方式构建在表达载体上，再转化到细菌、酵母、哺乳动物或昆虫细胞中，诱导表达出抗原蛋白，最后纯化并加入佐剂后制成疫苗。载体疫苗通过将特定病毒(如腺病毒、腺相关病毒等)作为遗传信息的载体，用基因工程技术将外源性抗原基因植入到病毒基因组内并转染细胞获得重组病毒，获得的重组病毒能在机体内利用宿主的遗传物质表达目的抗原蛋白，并诱导机体产生相应抗体，从而达到免疫接种的目的。核酸疫苗技术直接将编码抗原的核酸序列(DNA和mRNA)导入人体内，在人体细胞内合成蛋白质抗原，激发免疫应答。
联合疫苗是指含有两个或多个活的或灭活的生物体、或者提纯的抗原，用于预防多种疾病或由同一生物体的不同种或不同血清型引起的疾病。联合疫苗具有预防多种疾病、减少接种次数、简化免疫程序、提高接种率、降低交叉感染机会等优点，是当今疫苗研究的发展方向之一。联合疫苗指根据病原体的不同类型，而各型别之间交叉免疫性较低，针对不同型别病原体的设计，以预防多个型别感染的疫苗。一般是由同一制作方式制备出单价疫苗，再混合于同一针剂中供免疫使用。多联、多价疫苗是未来发展趋势，其优点在于：(1)节省疫苗接种的成本；(2)节省多个单个疫苗分别包装、物流和组装的成本；(3)减少对接种者，尤其是婴儿的伤害；(4)增加疫苗接种覆盖率；(5)节约疫苗接种时间；(6)减少疫苗储存空间等。联合或多价疫苗可以节省包括多次就诊、遗漏接种等在内的直接和间接成本等，如目前常用的多价联合疫苗包括肺炎链球菌、脑膜炎球菌、脊灰病毒、轮状病毒、流感、HPV等疫苗。
作为本发明所述疫苗组合物或多价疫苗组合物的优选实施方式，所述疫苗组合物或多价疫苗组合物还包括免疫学和药学上接受的载体或佐剂。
作为本发明所述疫苗组合物或多价疫苗组合物的优选实施方式，所述佐剂包括铝佐剂、弗氏佐剂、磷酸铝、磷酸钙、石蜡油、羊毛脂、表面活性剂、藻酸钙、多聚核苷酸、胞壁肽、皂苷、RIBI佐剂系统、霍乱毒素、丙烯酸或甲基丙烯酸的聚合物、油包水乳剂、水包油乳剂中的一种或几种，优选地，为铝佐剂。
本发明还提供了所述疫苗组合物或多价疫苗组合物在制备预防和/或治疗新型冠状病毒肺炎药物中的应用。
作为本发明所述的亚单位疫苗的优选实施方式，所述的预防和/或治疗是指在涉及SARS-CoV-2野生型和/或变异型毒株感染时抑制SARS-CoV-2野生型和/或变异型毒株的复制、传播或阻止其在宿主体内定居，以及减轻SARS-CoV-2野生型和/或变异型毒株感染的疾病或病症的症状。若使用亚单位疫苗后病毒荷载量降低和/或病症减轻就可以认为所述预防和/或治疗达到了效果。
本发明的有益效果：
(1)本发明设计制备的融合蛋白考察了不同RBD残基长度的免疫活性，并筛选最优的序列。
(2)本发明考察了融合蛋白中连接子的有无及连接子的种类对RBD体内免疫原性的影响。
(3)通过基因工程手段将蛋白RBD与人免疫球蛋白hFc域或His标签结合得到的融合蛋白RBD-hFc/RBD-His更便于提取与纯化，质量稳定可控，耗时短且可大规模生产。
(4)hFc/His融合蛋白技术可以增加RBD-hFc/RBD-His疫苗的溶解度、稳定性和在体内的半衰期。
(5)本发明的融合蛋白及其疫苗组合物可抑制SARS-CoV-2野生型和/或变异型毒株的复制、传播或阻止其在宿主体内定居，从而能有效预防和/或治疗SARS-CoV-2野生型和/或变异型毒株引起的新型冠状病毒肺炎。
附图说明
图1是本发明实施例1-3的融合蛋白疫苗诱导小鼠产生的抗体IgG免疫活性评价图；
图2是本发明实施例1-3的融合蛋白疫苗诱导小鼠产生的抗体IgG亚型免疫活性评价图；
图3是本发明实施例1-2与实施例4的融合蛋白不同给药剂量的亚单位疫苗诱导小鼠产生的抗体IgG亚型免疫活性评价图；
图4是本发明实施例1-2与实施例5的不同RBD-hFc的融合蛋白的亚单位疫苗诱导小鼠产生的抗体IgG亚型免疫活性评价图；
图5是本发明实施例1-2与实施例6的二价疫苗诱导小鼠产生的抗体IgG亚型免疫活性评价图；
图6是本发明实施例1-3的融合蛋白疫苗诱导小鼠产生的抗体中和实验免疫活性评价图；
图7是本发明实施例1-2与实施例4的融合蛋白不同给药剂量的亚单位疫苗诱导小鼠产生的抗体中和实验免疫活性评价图，HD-07的疫苗组中和抗体所用的毒株为野生毒株；
图8是本发明实施例1-2与实施例5的不同RBD-hFc的融合蛋白的亚单位疫苗诱导小鼠产生的抗体中和实验免疫活性评价图，含HD-02、HD-03与HD-08的疫苗组中和抗体所用的毒株为B.1.1.7变异型毒株，含HD-01、HD-06与HD-09的疫苗组中和抗体所用的毒株为501Y.V2变异型毒株，含HD-04、HD-05与HD-07的疫苗组中和抗体所用的毒株为野生毒株；
图9是本发明实施例1-2与实施例6的二价疫苗诱导小鼠产生的抗体中和实验免疫活性评价图，含HD-03的疫苗组中和抗体所用的毒株为B.1.1.7变异型毒株，含HD-05的疫苗组中和抗体所用的毒株为野生毒株；
图10是本发明实施例1-3的融合蛋白疫苗诱导小鼠产生的抗体血清特异性识别转染了ACE2载体的人肾上皮细胞系293T的流式细胞实验评价图；
图11是本发明实施例1-3的融合蛋白疫苗诱导小鼠脾细胞分泌出的IL-4细胞因子的免疫活性图，其中***P<0.001；
图12是本发明实施例1-3的融合蛋白疫苗诱导小鼠脾细胞分泌出的IFN-γ细胞因子的免疫活性图，其中***P<0.001。
具体实施方式
为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。
在本发明的描述中，需要说明的是，除非另有定义，本发明所使用的所有的技术和科学术语与属于本技术领域的技术人员通常理解的含义相同。本发明说明书中所使用的术语只是以描述具体的实施例为目的，不旨在限制本发明，对于本领域的普通技术人员而言，可以结合具体情况理解上述术语在本发明中的具体含义。
术语：融合蛋白(fusion protein)，有两种不同的含义，一种是通过DNA重组技术得到的两个基因重组后的表达产物，将两个不同的蛋白质连成一个大分子；另一种是通过化学方法连接两个不同的蛋白组成一个大分子。
本发明涉及的序列信息如下：
(1)SEQ ID No.1：野生型毒株S蛋白片段第319-593位氨基酸序列；
(2)SEQ ID No.2：野生型毒株的RBD(319-541)-hFc融合蛋白(编号HD-R1)人工合成核苷酸序列；
(3)SEQ ID No.3：野生型毒株的RBD(319-541)-hFc融合蛋白(编号HD-R1)人工合成氨基酸序列；
(4)SEQ ID No.4：野生型毒株的RBD(319-541)-His融合蛋白(编号HD-R2)人工合成核苷酸序列；
(5)SEQ ID No.5：野生型毒株的RBD(319-541)-His融合蛋白(编号HD-R2)人工合成氨基酸序列；
(6)SEQ ID No.6：野生型毒株的RBD(331-593)-hFc融合蛋白(编号HD-05)人工合成核苷酸序列；
(7)SEQ ID No.7：野生型毒株的RBD(331-593)-hFc融合蛋白(编号HD-05)人工合成氨基酸序列；
(8)SEQ ID No.8：野生型毒株的RBD(331-593)-His融合蛋白(编号HD-R3)人工合成核苷酸序列；
(9)SEQ ID No.9：野生型毒株的RBD(331-593)-His融合蛋白(编号HD-R3)人工合成氨基酸序列；
(10)SEQ ID No.10：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-541)-Fc融合蛋白(编号HD-01)核苷酸序列；
(11)SEQ ID No.11：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-541)-Fc融合蛋白(编号HD-01)氨基酸序列；
(12)SEQ ID No.12：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-541)-Fc融合蛋白(编号HD-02)人工合成核苷酸序列；
(13)SEQ ID No.13：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-541)-Fc融合蛋白(编号HD-02)人工合成氨基酸序列；
(14)SEQ ID No.14：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-Fc融合蛋白(编号HD-03)人工合成核苷酸序列；
(15)SEQ ID No.15：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-Fc融合蛋白(编号HD-03)人工合成氨基酸序列；
(16)SEQ ID No.16：野生型毒株的RBD(319-583)-hFc融合蛋白(编号HD-04)人工合成核苷酸序列；
(17)SEQ ID No.17：野生型毒株的RBD(319-583)-hFc融合蛋白(编号HD-04)人工合成氨基酸序列；
(18)SEQ ID No.18：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-583)-Fc融合蛋白(编号HD-06)人工合成核苷酸序列；
(19)SEQ ID No.19：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-583)-Fc融合蛋白(编号HD-06)人工合成氨基酸序列；
(20)SEQ ID No.20：野生型毒株的RBD(319-583)-hFc融合蛋白(编号HD-07)人工合成核苷酸序列；
(21)SEQ ID No.21：野生型毒株的RBD(319-583)-hFc融合蛋白(编号HD-07)人工合成氨基酸序列；
(22)SEQ ID No.22：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-Fc融合蛋白(编号HD-08)人工合成核苷酸序列；
(23)SEQ ID No.23：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-Fc融合蛋白(编号HD-08)人工合成氨基酸序列；
(24)SEQ ID No.24：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-583)-Fc融合蛋白(编号HD-09)人工合成核苷酸序列；
(25)SEQ ID No.25：501Y.V2变异型毒株(K417N,E484K,N501Y)的RBD(319-583)-Fc融合蛋白(编号HD-09)人工合成氨基酸序列；
(26)SEQ ID No.26：野生型毒株的RBD(319-583)-His融合蛋白(编号HD-10)人工合成核苷酸序列；
(27)SEQ ID No.27：野生型毒株的RBD(319-583)-His融合蛋白(编号HD-10)人工合成氨基酸序列；
(28)SEQ ID No.28：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-His融合蛋白(编号HD-11)人工合成核苷酸序列；
(29)SEQ ID No.29：B.1.1.7变异型毒株(N501Y，A570D)的RBD(319-583)-His融合蛋白(编号HD-11)人工合成氨基酸序列；
(30)SEQ ID No.30：501Y.V2变异型毒株(K417N，E484K，N501Y)的RBD(319-583)-His融合蛋白(编号HD-12)人工合成核苷酸序列；
(31)SEQ ID No.31：501Y.V2变异型毒株(K417N，E484K，N501Y)的RBD(319-583)-His融合蛋白(编号HD-12)人工合成氨基酸序列；
(32)SEQ ID No.32：蛋白hFc(编号HD-13)的核苷酸序列；
(33)SEQ ID No.33：蛋白hFc(编号HD-13)的氨基酸序列；
(34)SEQ ID No.34：野生型毒株的蛋白S1(1-685)-hFc融合蛋白(编号HD-S1)人工合成核苷酸序列；
(35)SEQ ID No.35：野生型毒株的蛋白S1(1-685)-hFc融合蛋白(编号HD-S1)人工合成氨基酸序列；
(36)SEQ ID No.36：野生型毒株的全长蛋白S(1-1213)-His融合蛋白(编号HD-S)人工合成核苷酸序列；
(37)SEQ ID No.37：野生型毒株的全长蛋白S(1-1213)-His融合蛋白(编号HD-S)人工合成核苷酸序列。
实施例1：融合蛋白HD-R1的制备、鉴定及含量测定
根据NCBI(https://www.ncbi.nlm.nih.gov)报道的新型冠状病毒中S蛋白片段(野生型毒株中S蛋白的GeneID：43740568)中RBD中和表位的基因序列(人工合成，氨基酸序列如SEQ ID No.1所示，为S蛋白氨基酸序列的第319-593位)，以及人免疫球蛋白IgG中Fc蛋白的基因序列(人工合成，核苷酸序列如SEQ ID No.32所示，氨基酸序列如SEQ ID No.33所示)，通过基因工程手段制备融合蛋白HD-R1(核苷酸序列如SEQ ID No.2所示，氨基酸序列如SEQ ID No.3所示)。
(1)PCDNA3.1-HD-R1重组表达质粒的构建及鉴定
在质粒载体pcDNA3.1 BamHI与XhoI酶切位点间插入双链DNA分子HD-R1，构建得到序列正确的重组表达质粒PCDNA3.1-RBD-hFc，测序鉴定正确。
(2)HD-R1蛋白在Expi293F悬浮型细胞中的表达
转染前，将3×106个Expi293F细胞接种于30mL Expi293F表达培养基中，持续培养数小时，当细胞密度达3.5×106个/mL时，将表达质粒加入5mL无血清的DMEM培养基中混匀，然后加入120μL Vortex Simple-Fect，混匀并室温孵育20分钟，将孵育好的体系缓慢加入细胞培养瓶中，并混匀，培养72小时后收集细胞进行HD-R1表达量的鉴定。
(3)HD-R1蛋白表达量的鉴定
取步骤(2)中的细胞培养液50μL，离心后用含有还原剂的蛋白上样缓冲液制样并电泳；电泳结束后，用湿转方法将蛋白转移到经甲醇激活的PVDF膜上；转膜后，用5％脱脂牛奶对PVDF膜室温摇床封闭1小时，分别用抗Fc兔多抗和Strep II-tag(HRP)抗体与封闭后的PVDF室温孵育1.5小时；用TBST清洗PVDF膜；加入羊抗兔二抗(HRP)室温孵育1小时；用TBST清洗PVDF膜；利用蛋白印迹显色液进行显色并鉴定HD-R1蛋白在Expi293F细胞中表达量。
(4)HD-R1蛋白的纯化及鉴定
利用Ni-NTA亲和柱对收获后的细胞总蛋白进行纯化，同时在纯化过程中收取目的蛋白的洗脱峰、柱前和柱后，进行SDS-PAGE分析，在收取的洗脱峰中获得了纯度较好的HD-R1蛋白。纯化后的HD-R1蛋白进行了蛋白印迹鉴定。
实施例2：其他融合蛋白的制备、鉴定及含量测定
表2中HD-R2、HD-05、HD-R3、HD-01、HD-02、HD-03、HD-04、HD-06、HD-07、HD-08、HD-09、HD-10、HD-11、HD-12、HD-S1和HD-S等16种融合蛋白的制备、鉴定及含量测定，除各自对应的质粒载体pcDNA3.1中分别含有其对应表2中16种融合蛋白氨基酸序列相对的核苷酸序列的不同外，其制备、鉴定及含量测定的步骤与实施例1类似。其中，16种融合蛋白是分别将不同RBD通过连接子与相应的标签相连接得到。
表2：17种融合蛋白的来源及特征信息
Figure GDA0003194726430000121
Figure GDA0003194726430000131
实施例3：亚单位疫苗的制备及小鼠免疫
(1)分别将实施例1和实施例2制备的融合蛋白HD-R1、HD-R2、HD-S1和HD-S用PBS缓冲盐溶液进行稀释，并加入铝佐剂(Al)充分乳化混匀即可得到相对应的亚单位疫苗，融合蛋白与铝佐剂的配比如表3所示。
表3：亚单位疫苗配比
Figure GDA0003194726430000141
(2)对于以上融合蛋白疫苗组和对照组，试验采用一次初始免疫(1st boost)和二次增强(2nd boost)免疫方案，小鼠分别在第0和30天肌肉注射制备的疫苗，每只每次注射量为10μg/100μL，对照组注射同体积的PBS溶液；第30、40天取血，每只小鼠取血0.1mL到0.2mL，在0℃放置60分钟，4000rpm离心15分钟，取上层清亮血清用于ELISA检测分析，并在第42天对小鼠进行安乐死，取脾脏制成单细胞混悬液用于后续ELISPOT实验。
实施例4：含有不同剂量HD-07融合蛋白的亚单位疫苗制备
用PBS缓冲盐溶液将实施例2制备的融合蛋白HD-07进行稀释，并加入铝佐剂(Al)充分乳化混匀，得到相对应的亚单位疫苗，HD-07亚单位疫苗配比如表4所示。
表4：融合蛋白不同剂量的亚单位疫苗配比
Figure GDA0003194726430000142
实施例5：HD-01、HD-02、HD-03、HD-04、HD-05、HD-06、HD-08和HD-09融合蛋白的亚单位疫苗制备
用PBS缓冲盐溶液将实施例2制备的融合蛋白HD-01、HD-02、HD-03、HD-04、HD-05、HD-06、HD-08和HD-09进行稀释，并加入铝佐剂(Al)充分乳化混匀，得到相对应亚单位疫苗，配比如表5所示。
表5：不同RBD-hFc的融合蛋白的亚单位疫苗配比
Figure GDA0003194726430000143
Figure GDA0003194726430000151
实施例6：二价疫苗的制备
分别将实施例1与实施例2中的融合蛋白HD-03、HD-05、HD-03+HD-R1和HD-05+HD-R1用PBS缓冲盐溶液进行稀释，并加入铝佐剂(Al)充分乳化混匀可得到相对应的亚单位疫苗，配比如表6所示。
表6：二价疫苗的配比
Figure GDA0003194726430000152
实施例7：小鼠分组免疫
取6-8周龄的Balb/c小鼠，随机分为16组，每组5只，将实施例4、实施例5和实施例6中的15种融合蛋白疫苗分别免疫小鼠，对照组Control(共1组，注射同体积的PBS溶液)，对于以上融合蛋白疫苗组和对照组，试验采用一次初始免疫(1st boost)和二次增强(2ndboost)免疫方案，分别在第0和14天肌肉注射制备的疫苗，每只每次注射量为100μL；第21天，每只小鼠取血0.1mL到0.2mL，在0℃放置60分钟，4000rpm离心15分钟，取上层清亮血清用于ELISA检测和中和抗体分析。
实施例8：ELISA免疫分析
(1)用0.1M碳酸盐缓冲液(pH 9.6)将蛋白抗原【野生型毒株铺板蛋白为HD-10(野生型)，B.1.1.7变异型毒株铺板蛋白为HD-11(B.1.1.7突变株)，501Y.V2变异型毒株铺板蛋白为HD-12(501Y.V2突变株)】配置成浓度为0.5μg/ml的溶液，以每孔100μL的量加入96孔板，放入4℃孵育过夜；第二天放入37℃培养箱孵育1小时；用PBST(PBS+0.1％吐温20)洗板3次，每孔加入300μL洗液；洗板后加入2％脱脂奶粉，每孔加入250μL，37℃孵育1小时；用PBST(PBS+0.1％吐温20)洗板3次。分别在每个孔中加入hFc标记的融合蛋白疫苗免疫后的小鼠血清(稀释倍数1:300)，每孔加入100μL，放置在37℃培养箱孵育1小时；洗板3次后；分别在一次免疫或二次免疫的小鼠血清组加入HRP(辣根过氧化物酶)标记的IgG，在二次免疫的小鼠血清组加入HRP(辣根过氧化物酶)标记的IgG1、IgG2b、IgG2c与IgG3，每孔加入100μL，室温孵育1小时；洗板3次。加入TMB溶液，每孔加入100μL，室温避光显色15分钟。加入0.5MH2SO4溶液终止显色，每孔加100μL，酶标仪检测吸光度，检测波长为450nm，570nm作为背景波长。
(2)吸光度结果分析
1)将吸光度(OD)值相对于实施例3中不同亚单位疫苗作图，如图1所示，本发明实施例3中的亚单位融合蛋白疫苗HD-R1/PBS及其疫苗组合物HD-R1/Al、HD-R2/Al、HD-S1/Al与HD-S/Al，在小鼠体内均能诱导产生高滴度的IgG抗体，第二次免疫后的OD值较第一次免疫有显著升高，表明小鼠首次疫苗免疫后再次接受同样的疫苗抗原能迅速并大量产生特异性抗体；其中RBD-hFc(HD-R1/Al与HD-R1/PBS)的融合蛋白疫苗产生抗体的OD值显著高于S1-hFc(HD-S1/Al)和S-His(HD-S/Al)的融合蛋白疫苗，说明RBD作为融合蛋白抗原的效果比S1与全长S蛋白好；RBD-hFc(HD-R1/Al与HD-R1/PBS)的融合蛋白疫苗产生的抗体的OD值均明显高于RBD-His(HD-R2/Al)的融合蛋白疫苗，说明hFc能有效提高RBD的免疫原性；HD-R1/Al组的OD值高于HD-R1/PBS组，表明铝佐剂能增强RBD-hFc的免疫原性。
2)将吸光度(OD)值相对于实施例3中不同亚型抗体作图，如图2所示，亚单位融合蛋白疫苗HD-R1/PBS及其疫苗组合物HD-R1/Al、HD-R2/Al和HD-S/Al组免疫的小鼠显著增强了IgG1和IgG2b的分泌，表明RBD的融合蛋白疫苗可以同时诱导Th2和Th1免疫反应并产生强效的抗体，RBD-hFc(HD-R1/Al、HD-R1/PBS)组诱导分泌的IgG1和IgG2b的OD值明显高于S-hFc(HD-S/Al)组与RBD-His(HD-R2/Al)组，再次验证了RBD作为融合蛋白抗原的效果比S1好，hFc能有效提高RBD的免疫原性。
3)将吸光度(OD)值相对于实施例4中融合蛋白不同给药剂量的疫苗组合物HD-07/Al作图，如图3所示，三组不同蛋白剂量的疫苗诱导小鼠产生的IgG抗体值大小如下：10μgHD-07/100μg Al>5μgHD-07/100μg Al>15μgHD-07/100μg Al，说明该融合蛋白作为疫苗给药10μg时效果最好。
4)将吸光度(OD)值相对于实施例5中不同亚单位疫苗作图，如图4所示，本发明实施例6中的亚单位融合蛋白疫苗组合物10μg HD-01/100μg Al～10μg HD-09/100μg Al，在小鼠体内诱导产生的IgG抗体的OD值均显著高于空白组，说明该融合蛋白均能诱导有效的免疫反应，可作为疫苗中的抗原；其他条件一样的情况下，以AAA作为融合蛋白连接子的疫苗组合物10μg HD-07/100μg Al诱导产生抗体的OD值显著高于以ADDDDK作为连接子的10μgHD-04/100μg Al，501Y.V2变异型毒株组以AAA作为融合蛋白连接子的10μg HD-09/100μgAl诱导产生抗体的OD值也比HD-06/Al高，说明作为融合蛋白的连接子，AAA比ADDDDK更能增强RBD的免疫原性，融合蛋白中的连接子影响融合蛋白抗原的免疫原性。
5)将吸光度(OD)值相对实施例6中的二价疫苗组合物作图，如图5所示，二价疫苗10μg HD-03+10μg HD-R1/1mg Al与10μg HD-05+10μg HD-R1/1mg Al在小鼠体内诱导产生IgG抗体的OD值显著高于单价疫苗10μg HD-03/1mg Al与10μg HD-05/1mg Al，说明二价疫苗中变异型毒株的RBD作为抗原诱导产生的抗体也能识别野生型毒株的RBD，即变异型毒株产生的抗体与野生型毒株产生的抗体能交叉识别对方的抗原。
实施例9：中和抗体实验
将Vero E6细胞(2×104个/孔)接种到96孔板中，在37℃和5％CO2下培养过夜，直到形成单层。将SARS-CoV-2(HD-02、HD-03与HD-08的疫苗组用B.1.1.7变异型毒株，HD-01、HD-06与HD-09的疫苗组用501Y.V2变异型毒株，HD-04、HD-05与HD-07的疫苗组用野生毒株)的100TCID50与连续4倍稀释的小鼠抗血清混合，在37℃孵育一小时，将血清样品在56℃加热30分钟，随后加入到Vero E6细胞培养孔混合。在每种测定中，感染了100TCID50SARS-CoV-2的细胞为阳性对照，而无病毒的细胞为阴性对照，然后在感染后第3天记录CPE(细胞病变效应)。通过Reed-Muench法计数SARS-CoV-2病毒感染Vero E6细胞数(CPE)，能使50％接种后的细胞产生细胞病变小鼠血清的最大稀释倍数，即为此疫苗的中和抗体滴度(NA)。
中和抗体滴度越高，说明病毒复制水平越低，对病毒感染的防护能力越强。将中和抗体(NA)值相对于实施例3中的不同融合蛋白作图，如图6所示，HD-R1/PBS、HD-R1/Al与HD-S1/Al组在第二次免疫后诱导的血清中和抗体滴度显著升高，表明S1-hFc组(HD-S/Al)与RBD-hFc组(HD-R1/Al、HD-R1/PBS)均能产生强效的体液免疫应答，其诱导产生的抗体血清具有较强的活性，并保护细胞免受SARS-CoV-2感染；RBD-hFc组(HD-R1/Al、HD-R1/PBS)中和抗体滴度显著高于S1-hFc组(HD-S/Al)组，说明RBD比S1和S蛋白更具有作为疫苗特异性抗原的潜力；没有使用hFc的RBD-His组(HD-R2/Al)和全长蛋白S-His组(HD-S/Al)产生的中和抗体滴度接近于0，再次说明hFc可以有效提高RBD的免疫原性。
将中和抗体(NA)值相对于实施例4融合蛋白不同给药剂量的疫苗组合物HD-07/Al作图，如图7所示，10μg HD-07/100μg Al组疫苗诱导小鼠产生的血清中和抗体值显著高于15μg HD-07/100μg Al与5μg HD-07/100μg Al，说明该融合蛋白作为疫苗给药10μg时效果最好。
将中和抗体(NA)值实施例5中相对于不同亚单位疫苗作图，如图8所示，各组疫苗组合物给药后第21天诱导的抗体血清的中和抗体滴度显著高于空白组疫苗，表明以突变株的RBD为抗原的疫苗诱导产生的抗体血清具有较强的中和活性，并保护细胞免受SARS-CoV-2感染。在其他条件一样的情况下，以AAA作为融合蛋白连接子的疫苗组合物10μg HD-07/100μg Al诱导产生中和抗体滴度显著高于以ADDDDK作为连接子的10μg HD-04/100μg Al，501Y.V2变异型毒株组以AAA作为融合蛋白连接子的HD-09/Al诱导产生的中和抗体滴度高于10μg HD-06/100μg Al，以及N501Y变异型毒株组以AAA作为融合蛋白连接子的10μg HD-08/100μg Al诱导产生中和抗体滴度也比10μg HD-03/100μg Al高，说明作为融合蛋白的连接子AAA比ADDDDK更能增强RBD的中和抗体活性，抗病毒感染能力更强。
将中和抗体(NA)值相对于实施例6中的二价疫苗组合物作图，如图9所示，二价疫苗10μg HD-03+10μgHD-R1/1mg Al与10μg HD-05+10μg HD-R1/1mg Al在小鼠体内均能诱导产生高滴度的血清中和抗体值，说明野生型毒株与变异型毒株联合给药产生的抗体均能中和病毒感染，其中二价疫苗10μgHD-03+10μg HD-R1/1mg Al产生的血清中和抗体值显著高于单价疫苗10μgHD-03/100μg Al，说明野生型毒株诱导产生的抗体可以中和变异型毒株。
实施例10：流式细胞实验
将人肾上皮细胞系293T用人ACE2载体转染48h，分离并用PBS洗涤细胞3次，计数使293T/ACE2 1.0×106个细胞/管，将采用hFc标记的融合蛋白疫苗免疫后小鼠的血清(1:10稀释)分别加入到293T/ACE2细胞中，然后加入1μg/mL RBD-His蛋白，在冰上孵育1h。用PBS洗涤3次后，加入anti-His-FITC，在冰上避光孵育1h，用PBS洗涤3次后通过流式细胞仪检测平均荧光强度。
将人肾上皮细胞系293T用人ACE2载体转染48h，分离并用PBS洗涤细胞3次，计数使293T/ACE2 1.0×106个细胞/管，将采用His标记的融合蛋白疫苗免疫后小鼠的血清(1:10稀释)分别加入到293T/ACE2细胞中，然后加入1μg/mL RBD-hFc蛋白，在冰上孵育1h。用PBS洗涤3次后，加入anti-hFc-FITC，在冰上避光孵育1h，用PBS洗涤3次后通过流式细胞仪检测平均荧光强度。
结果如图10所示，与Positive control(阳性对照)组相比，亚单位疫苗HD-R1/Al(RBD-hFc)的峰图明显向左移，与Negative control(阴性对照)组几乎重合，PositiveControl(空白血清)组峰图明显右移，计算得到抑制率为67.33％，说明亚单位疫苗HD-R1/Al组(RBD-hFc)的小鼠血清在1:10稀释时有效阻断了RBD与293T/ACE2细胞的结合，而对照的空白血清在相同稀释度下没有抑制作用。亚单位疫苗HD-R2/Al(RBD-His)诱导的小鼠血清的荧光峰在Negative control(阴性对照)组与Positive Control(空白血清)组荧光峰之间，计算得到抑制率为36.75％，说明HD-R2/Al(RBD-His)诱导的小鼠血清有效阻断了RBD与293T/ACE2细胞的结合，但其抑制效果没有HD-R1/Al(RBD-hFc)好，再次验证了hFc组有效提高RBD的免疫原性。
实施例11：Elispot实验
第一天接种细胞：无菌条件下分离小鼠脾脏，于6孔板中加入淋巴细胞分离液，4mL/孔，将单只小鼠脾脏置于200目尼龙网细胞筛，用5mL注射器芯研磨为细胞悬液，将细胞悬液转移至15mL离心管中，缓缓加入1mL无血清的1640培养基，浮于细胞悬液上方；800rpm离心30min后，用移液枪插至白膜层，吸取尽量多的淋巴细胞加到含有5mL无血清的1640培养基15mL离心管中。用PBS洗涤细胞3次，800rpm水平离心10min，弃上清，每管加入500μL无血清的1640培养基重悬后进行细胞计数，调整细胞密度为1×106个/mL。
每孔加入200μL无血清的1640培养基，室温静置5-10分钟后将其取出，活化预包被板；将调整好浓度的细胞悬液加入各实验孔，100μL/孔；加入刺激物：10μL/孔，所有样品和刺激物加完后，盖好板盖，放入37℃，5％CO2培养箱培养16-24小时。
第二天倾倒孔内细胞及培养基，加入冰冷的去离子水，200μL/孔，4℃冰箱放置10分钟，低渗裂解细胞，甩出孔内液体，每孔加入260μL 1×Washing buffer/孔洗5次；将稀释好的生物素标记的抗体(Biotinylated antibody)工作液加入各实验孔，100μL/孔，37℃孵育1小时；洗板5次；将稀释好的酶标亲和素(Streptavidin-HRP)工作液加入各实验孔，100μL/孔，37℃孵育1小时；洗板5次；将现配的AEC显色液加入各实验孔，100μL/孔；室温避光静置5-30分钟，根据斑点生成情况选择终止显色时间；倾倒孔内液体，揭开板底座，用去离子水洗涤正反面及底座3-5遍，终止显色，将板放置在室温阴凉处，待其自然晾干后上机检测斑点数。
结果如图11、图12所示，从免疫后小鼠中分离出的脾细胞分泌出的IL-4及IFN-γ数量多少关系为：HD-R1/Al组>HD-R1/PBS组>HD-R2/Al组，HD-R2/Al组分泌的IFN-γ较少，接近空白组，表明RBD-hFc(HD-R1/Al及HD-R1/PBS)融合疫苗可同时诱导体液免疫和细胞免疫应答，其免疫效果均比(RBD-His)HD-R2/Al好，说明hFc可有效提高RBD在体内的免疫应答能力；HD-R1/Al组脾细胞分泌出的IL-4及IFN-γ均高于HD-R1/Al组，表明铝佐剂能增强RBD-hFc的免疫原性(***P<0.001，表示差异性显著)。
综上所述，以RBD蛋白与人免疫球蛋白hFc域或His标签结合得到融合蛋白制成的亚单位疫苗RBD-hFc与RBD-His，及在该融合蛋白中加入佐剂制成的疫苗组合物，可以促进其被选择性吸收，并诱导针对SARS-CoV-2的特异性免疫反应，达到抑制SARS-CoV-2野生型和/或变异型毒株的复制、传播或阻止其在宿主体内定居的目的。免疫活性评价中，以RBD作为抗原的融合蛋白疫苗诱导的免疫活性均明显比S1和S强，因此，筛选出RBD作为疫苗最理想的特异性抗原。RBD-hFc(HD-R1/Al、HD-R1/PBS)融合蛋白诱导产生的抗体及抗体特异性识别ACE2的能力均显著高于RBD-His(HD-R2/Al)，说明hFc可以有效提高RBD的免疫原性。HD-R1/Al组较HD-R1/PBS组免疫活性强，表明铝佐剂能有效提高融合蛋白的免疫原性。在不同剂量给药的研究中，融合蛋白的剂量为10μg时最佳，过低或过高均影响其免疫效果；此外，融合蛋白中的连接子与RBD长度影响抗原的免疫原性。二价疫苗HD-03+HD-R1/1mg Al与HD-05+HD-R1/1mg Al在小鼠体内均能诱导产生IgG抗体的OD值显著高于单价疫苗HD-03/1mg与HD-05/1mg Al，说明二价疫苗对野生型毒株和/或变异型毒株同时都具有免疫反应作用。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 广东克冠达生物医药科技有限公司
<120> 一种COVID-19亚单位疫苗及其制备方法与应用
<130> 2021.04.16
<150> US63145505
<151> 2021-02-04
<160> 37
<170> PatentIn version 3.3
<210> 1
<211> 275
<212> PRT
<213> SARS-CoV-2
<400> 1
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys
260 265 270
Ser Phe Gly
275
<210> 2
<211> 1371
<212> DNA
<213> 人工合成
<400> 2
agggtgcagc ctaccgagag catcgtgaga ttccctaata tcaccaacct gtgtcccttc 60
ggcgaggtgt tcaatgccac cagatttgcc agcgtgtacg cctggaacag gaagagaatc 120
agcaactgcg tggccgacta ctccgtgctg tacaactccg ccagcttcag cacctttaag 180
tgctacggcg tgagccccac caagctgaac gatctgtgct tcacaaatgt gtacgccgat 240
agcttcgtga tcagaggcga cgaggtgagg cagatcgccc ctggccagac aggcaagatc 300
gccgattaca actacaagct gcccgatgac ttcacaggct gcgtgatcgc ctggaatagc 360
aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaacctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccctgtaatg gcgtggaggg ctttaactgc tactttcccc tgcagtccta cggctttcag 540
cctaccaatg gcgtgggcta ccagccctac agagtggtgg tgctgtcctt cgagctgctg 600
cacgcccccg ccaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgt 660
gtgaattttg ccgacgacga cgataaggcc gtgcctagag atagcggctg taagccttgt 720
atctgtacag tgcctgaggt gtccagcgtg tttatcttcc cccccaagcc caaggatgtg 780
ctgacaatca ccctgacccc caaggtgaca tgcgtggtgg tggatatctc caaggacgac 840
cccgaggtgc agttctcctg gttcgtggac gatgtggagg tgcacacagc ccagacacag 900
cctagggagg agcagttcaa ctccaccttt agaagcgtga gcgagctgcc catcatgcac 960
caggactggc tgaacggcaa ggagtttaag tgtagggtga atagcgccgc cttccccgcc 1020
cctatcgaga agaccatcag caagacaaag ggcagaccca aggcccctca ggtgtacacc 1080
atccctcccc ctaaggagca gatggccaag gacaaggtga gcctgacatg tatgatcaca 1140
gatttctttc ccgaggacat cacagtggag tggcagtgga atggccagcc cgccgagaac 1200
tacaagaata cccagcccat catggacacc gacggctcct acttcgtgta cagcaagctg 1260
aacgtgcaga agagcaactg ggaggccggc aacaccttta cctgcagcgt gctgcacgag 1320
ggcctgcaca accaccacac cgagaagagc ctgtcccaca gccctggcaa g 1371
<210> 3
<211> 461
<212> PRT
<213> 人工合成
<400> 3
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala
210 215 220
Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 4
<211> 702
<212> DNA
<213> 人工合成
<400> 4
agggttcagc ccacagagag catcgtgagg ttccccaata tcacaaacct gtgccctttt 60
ggcgaggtgt tcaatgccac caggttcgcc agcgtgtacg cctggaacag aaagaggatc 120
tccaattgcg tggccgatta ctccgtgctg tacaattccg cctccttcag cacattcaaa 180
tgctacggcg tgagccccac aaagctgaat gatctgtgct ttaccaatgt gtacgccgat 240
tcctttgtga tcagaggcga cgaggtgaga cagatcgccc caggacagac cggaaagatc 300
gccgattaca actacaagct gcctgacgac ttcaccggct gcgtgatcgc ttggaacagc 360
aacaacctgg atagcaaggt gggcggcaat tacaattacc tgtacaggct gttcagaaag 420
tccaatctga agcccttcga gagggacatc tccacagaga tctaccaggc cggcagcacc 480
ccatgtaacg gagttgaggg cttcaactgt tacttccccc tgcagtccta cggcttccag 540
cctacaaatg gcgtgggcta ccagccttac agggtggttg tgctgagctt cgagctgctg 600
cacgcccctg ctacagtgtg tggaccaaag aagagcacca acctggtgaa gaacaagtgt 660
gtgaactttg cccaccacca ccaccatcac caccaccatc at 702
<210> 5
<211> 234
<212> PRT
<213> 人工合成
<400> 5
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala
210 215 220
His His His His His His His His His His
225 230
<210> 6
<211> 1485
<212> DNA
<213> 人工合成
<400> 6
aatatcacaa acctgtgccc ttttggcgag gtgttcaatg ccaccaggtt cgccagcgtg 60
tacgcctgga acagaaagag gatctccaat tgcgtggccg attactccgt gctgtacaat 120
tccgcctcct tcagcacatt caaatgctac ggcgtgagcc ccacaaagct gaatgatctg 180
tgctttacca atgtgtacgc cgattccttt gtgatcagag gcgacgaggt gagacagatc 240
gccccaggac agaccggaaa gatcgccgat tacaactaca agctgcctga cgacttcacc 300
ggctgcgtga tcgcttggaa cagcaacaac ctggatagca aggtgggcgg caattacaat 360
tacctgtaca ggctgttcag aaagtccaat ctgaagccct tcgagaggga catctccaca 420
gagatctacc aggccggcag caccccatgt aacggagttg agggcttcaa ctgttacttc 480
cccctgcagt cctacggctt ccagcctaca aatggcgtgg gctaccagcc ttacagggtg 540
gttgtgctga gcttcgagct gctgcacgcc cctgctacag tgtgtggacc aaagaagagc 600
accaacctgg tgaagaacaa gtgtgtgaac tttaacttca acggcctgac cggcaccgga 660
gtgctgacag aatccaataa gaagtttctg ccctttcagc agttcggcag agatatcgcc 720
gacacaacag atgccgtgag ggaccctcag accctggaaa tcctggacat caccccctgc 780
agctttggcg agcctaagtc ctccgataag acacacacct gccccccttg tcccgcccct 840
gagctgctgg gaggcccatc tgtgttcctg tttcccccta agcctaagga taccctgatg 900
atcagcagaa cacccgaggt gacctgcgtg gtggtggatg tgtcccacga ggaccctgag 960
gtgaagttca attggtacgt ggatggcgtg gaggtgcaca atgccaagac aaagcctagg 1020
gaggagcagt acaattccac atacagagtg gtgagcgtgc tgaccgtgct gcaccaggat 1080
tggctgaacg gcaaggagta caagtgcaag gtgagcaata aggccctgcc cgcccctatc 1140
gagaagacca tcagcaaggc caagggccag cccagggagc ctcaggtgta cacactgcct 1200
ccttccagag acgagctgac caagaaccag gtgtccctga cctgtctggt gaaaggcttc 1260
tacccctccg acatcgccgt ggagtgggag tccaatggcc agcctgagaa caactacaag 1320
accacacctc ccgtgctgga tagcgatggc tcctttttcc tgtacagcaa gctgacagtg 1380
gacaagtcca ggtggcagca gggcaatgtg tttagctgct ccgtgatgca cgaggccctg 1440
cacaaccact acacccagaa gagcctgtcc ctgtcccccg gcaag 1485
<210> 7
<211> 495
<212> PRT
<213> 人工合成
<400> 7
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
1 5 10 15
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
100 105 110
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
115 120 125
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
130 135 140
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
145 150 155 160
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
165 170 175
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
180 185 190
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
195 200 205
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu
210 215 220
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
225 230 235 240
Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp
245 250 255
Ile Thr Pro Cys Ser Phe Gly Glu Pro Lys Ser Ser Asp Lys Thr His
260 265 270
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
275 280 285
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
290 295 300
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
305 310 315 320
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
325 330 335
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
340 345 350
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
355 360 365
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
370 375 380
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
385 390 395 400
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
405 410 415
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
420 425 430
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
435 440 445
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
450 455 460
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
465 470 475 480
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
485 490 495
<210> 8
<211> 819
<212> DNA
<213> 人工合成
<400> 8
aatatcacaa acctgtgccc ttttggcgag gtgttcaatg ccaccaggtt cgccagcgtg 60
tacgcctgga acagaaagag gatctccaat tgcgtggccg attactccgt gctgtacaat 120
tccgcctcct tcagcacatt caaatgctac ggcgtgagcc ccacaaagct gaatgatctg 180
tgctttacca atgtgtacgc cgattccttt gtgatcagag gcgacgaggt gagacagatc 240
gccccaggac agaccggaaa gatcgccgat tacaactaca agctgcctga cgacttcacc 300
ggctgcgtga tcgcttggaa cagcaacaac ctggatagca aggtgggcgg caattacaat 360
tacctgtaca ggctgttcag aaagtccaat ctgaagccct tcgagaggga catctccaca 420
gagatctacc aggccggcag caccccatgt aacggagttg agggcttcaa ctgttacttc 480
cccctgcagt cctacggctt ccagcctaca aatggcgtgg gctaccagcc ttacagggtg 540
gttgtgctga gcttcgagct gctgcacgcc cctgctacag tgtgtggacc aaagaagagc 600
accaacctgg tgaagaacaa gtgtgtgaac tttaacttca acggcctgac cggcaccgga 660
gtgctgacag aatccaataa gaagtttctg ccctttcagc agttcggcag agatatcgcc 720
gacacaacag atgccgtgag ggaccctcag accctggaaa tcctggacat caccccctgc 780
agctttggcc accaccacca ccatcaccac caccatcat 819
<210> 9
<211> 273
<212> PRT
<213> 人工合成
<400> 9
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
1 5 10 15
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
100 105 110
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
115 120 125
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
130 135 140
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
145 150 155 160
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
165 170 175
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
180 185 190
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
195 200 205
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu
210 215 220
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
225 230 235 240
Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp
245 250 255
Ile Thr Pro Cys Ser Phe Gly His His His His His His His His His
260 265 270
His
<210> 10
<211> 1383
<212> DNA
<213> 人工合成
<400> 10
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaatatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttaaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaactttg ccgacgacga tgacaaggag cccaagtcca gcgataagac acacacctgc 720
cctccttgtc ccgcccctga actgctggga ggaccttctg tgttcctgtt cccccccaag 780
cctaaggaca ccctgatgat cagcagaacc cccgaggtga cctgtgtggt ggtggatgtg 840
tcccacgagg acccagaggt gaagttcaac tggtacgtgg atggcgtgga ggtgcacaac 900
gccaagacaa agcctaggga ggagcagtac aacagcacat acagagtggt ctccgtgctg 960
acagtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gtccaataag 1020
gccctgcctg cccctatcga gaagaccatc agcaaggcca agggccagcc tagagagcct 1080
caggtttaca cactgccccc ttccagggac gagctgacaa agaaccaggt gtccctgacc 1140
tgcctggtga agggattcta ccctagcgac atcgccgtgg agtgggagtc taatggccag 1200
ccagagaata actacaagac aacccccccc gtgctggatt ccgatggatc tttctttctg 1260
tacagcaagc tgacagtgga taagtccagg tggcagcagg gcaacgtgtt ttcctgtagc 1320
gtgatgcacg aggccctgca caatcactac acccagaaga gcctgagcct gagccctgga 1380
aag 1383
<210> 11
<211> 461
<212> PRT
<213> 人工合成
<400> 11
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala
210 215 220
Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 12
<211> 1383
<212> DNA
<213> 人工合成
<400> 12
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaactttg ccgacgacga tgacaaggag cccaagtcca gcgataagac acacacctgc 720
cctccttgtc ccgcccctga actgctggga ggaccttctg tgttcctgtt cccccccaag 780
cctaaggaca ccctgatgat cagcagaacc cccgaggtga cctgtgtggt ggtggatgtg 840
tcccacgagg acccagaggt gaagttcaac tggtacgtgg atggcgtgga ggtgcacaac 900
gccaagacaa agcctaggga ggagcagtac aacagcacat acagagtggt ctccgtgctg 960
acagtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gtccaataag 1020
gccctgcctg cccctatcga gaagaccatc agcaaggcca agggccagcc tagagagcct 1080
caggtttaca cactgccccc ttccagggac gagctgacaa agaaccaggt gtccctgacc 1140
tgcctggtga agggattcta ccctagcgac atcgccgtgg agtgggagtc taatggccag 1200
ccagagaata actacaagac aacccccccc gtgctggatt ccgatggatc tttctttctg 1260
tacagcaagc tgacagtgga taagtccagg tggcagcagg gcaacgtgtt ttcctgtagc 1320
gtgatgcacg aggccctgca caatcactac acccagaaga gcctgagcct gagccctgga 1380
aag 1383
<210> 13
<211> 461
<212> PRT
<213> 人工合成
<400> 13
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala
210 215 220
Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 14
<211> 1509
<212> DNA
<213> 人工合成
<400> 14
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgaccggc accggagtgc tgacagaatc caataagaag 720
ttcctgccct ttcagcagtt cggcagagac atcgatgata ccaccgacgc cgtgagagac 780
cctcagacac tggaggccga cgacgatgac aaggagccca agtccagcga taagacacac 840
acctgccctc cttgtcccgc ccctgaactg ctgggaggac cttctgtgtt cctgttcccc 900
cccaagccta aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 960
gatgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggatgg cgtggaggtg 1020
cacaacgcca agacaaagcc tagggaggag cagtacaaca gcacatacag agtggtctcc 1080
gtgctgacag tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 1140
aataaggccc tgcctgcccc tatcgagaag accatcagca aggccaaggg ccagcctaga 1200
gagcctcagg tttacacact gcccccttcc agggacgagc tgacaaagaa ccaggtgtcc 1260
ctgacctgcc tggtgaaggg attctaccct agcgacatcg ccgtggagtg ggagtctaat 1320
ggccagccag agaataacta caagacaacc ccccccgtgc tggattccga tggatctttc 1380
tttctgtaca gcaagctgac agtggataag tccaggtggc agcagggcaa cgtgttttcc 1440
tgtagcgtga tgcacgaggc cctgcacaat cactacaccc agaagagcct gagcctgagc 1500
cctggaaag 1509
<210> 15
<211> 503
<212> PRT
<213> 人工合成
<400> 15
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Asp Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Asp Asp Asp Asp Lys Glu
260 265 270
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
275 280 285
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
290 295 300
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
305 310 315 320
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
325 330 335
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
340 345 350
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
355 360 365
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
370 375 380
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
385 390 395 400
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
405 410 415
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
420 425 430
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
435 440 445
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
450 455 460
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
465 470 475 480
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
485 490 495
Leu Ser Leu Ser Pro Gly Lys
500
<210> 16
<211> 1509
<212> DNA
<213> 人工合成
<400> 16
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggaggccga cgacgatgac aaggagccca agtccagcga taagacacac 840
acctgccctc cttgtcccgc ccctgaactg ctgggaggac cttctgtgtt cctgttcccc 900
cccaagccta aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 960
gatgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggatgg cgtggaggtg 1020
cacaacgcca agacaaagcc tagggaggag cagtacaaca gcacatacag agtggtctcc 1080
gtgctgacag tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 1140
aataaggccc tgcctgcccc tatcgagaag accatcagca aggccaaggg ccagcctaga 1200
gagcctcagg tttacacact gcccccttcc agggacgagc tgacaaagaa ccaggtgtcc 1260
ctgacctgcc tggtgaaggg attctaccct agcgacatcg ccgtggagtg ggagtctaat 1320
ggccagccag agaataacta caagacaacc ccccccgtgc tggattccga tggatctttc 1380
tttctgtaca gcaagctgac agtggataag tccaggtggc agcagggcaa cgtgttttcc 1440
tgtagcgtga tgcacgaggc cctgcacaat cactacaccc agaagagcct gagcctgagc 1500
cctggaaag 1509
<210> 17
<211> 503
<212> PRT
<213> 人工合成
<400> 17
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Asp Asp Asp Asp Lys Glu
260 265 270
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
275 280 285
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
290 295 300
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
305 310 315 320
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
325 330 335
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
340 345 350
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
355 360 365
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
370 375 380
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
385 390 395 400
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
405 410 415
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
420 425 430
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
435 440 445
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
450 455 460
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
465 470 475 480
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
485 490 495
Leu Ser Leu Ser Pro Gly Lys
500
<210> 18
<211> 1509
<212> DNA
<213> 人工合成
<400> 18
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaatatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttaaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggaggccga cgacgatgac aaggagccca agtccagcga taagacacac 840
acctgccctc cttgtcccgc ccctgaactg ctgggaggac cttctgtgtt cctgttcccc 900
cccaagccta aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 960
gatgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggatgg cgtggaggtg 1020
cacaacgcca agacaaagcc tagggaggag cagtacaaca gcacatacag agtggtctcc 1080
gtgctgacag tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 1140
aataaggccc tgcctgcccc tatcgagaag accatcagca aggccaaggg ccagcctaga 1200
gagcctcagg tttacacact gcccccttcc agggacgagc tgacaaagaa ccaggtgtcc 1260
ctgacctgcc tggtgaaggg attctaccct agcgacatcg ccgtggagtg ggagtctaat 1320
ggccagccag agaataacta caagacaacc ccccccgtgc tggattccga tggatctttc 1380
tttctgtaca gcaagctgac agtggataag tccaggtggc agcagggcaa cgtgttttcc 1440
tgtagcgtga tgcacgaggc cctgcacaat cactacaccc agaagagcct gagcctgagc 1500
cctggaaag 1509
<210> 19
<211> 503
<212> PRT
<213> 人工合成
<400> 19
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Asp Asp Asp Asp Lys Glu
260 265 270
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
275 280 285
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
290 295 300
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
305 310 315 320
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
325 330 335
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
340 345 350
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
355 360 365
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
370 375 380
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
385 390 395 400
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
405 410 415
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
420 425 430
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
435 440 445
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
450 455 460
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
465 470 475 480
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
485 490 495
Leu Ser Leu Ser Pro Gly Lys
500
<210> 20
<211> 1500
<212> DNA
<213> 人工合成
<400> 20
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggaggccgc cgccgagccc aagtccagcg ataagacaca cacctgccct 840
ccttgtcccg cccctgaact gctgggagga ccttctgtgt tcctgttccc ccccaagcct 900
aaggacaccc tgatgatcag cagaaccccc gaggtgacct gtgtggtggt ggatgtgtcc 960
cacgaggacc cagaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaacgcc 1020
aagacaaagc ctagggagga gcagtacaac agcacataca gagtggtctc cgtgctgaca 1080
gtgctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtgtc caataaggcc 1140
ctgcctgccc ctatcgagaa gaccatcagc aaggccaagg gccagcctag agagcctcag 1200
gtttacacac tgcccccttc cagggacgag ctgacaaaga accaggtgtc cctgacctgc 1260
ctggtgaagg gattctaccc tagcgacatc gccgtggagt gggagtctaa tggccagcca 1320
gagaataact acaagacaac cccccccgtg ctggattccg atggatcttt ctttctgtac 1380
agcaagctga cagtggataa gtccaggtgg cagcagggca acgtgttttc ctgtagcgtg 1440
atgcacgagg ccctgcacaa tcactacacc cagaagagcc tgagcctgag ccctggaaag 1500
<210> 21
<211> 500
<212> PRT
<213> 人工合成
<400> 21
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Ala Ala Glu Pro Lys Ser
260 265 270
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
275 280 285
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440 445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Pro Gly Lys
500
<210> 22
<211> 1500
<212> DNA
<213> 人工合成
<400> 22
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgacgaca caacagacgc tgtgagagat 780
cctcagaccc tggaggccgc cgccgagccc aagtccagcg ataagacaca cacctgccct 840
ccttgtcccg cccctgaact gctgggagga ccttctgtgt tcctgttccc ccccaagcct 900
aaggacaccc tgatgatcag cagaaccccc gaggtgacct gtgtggtggt ggatgtgtcc 960
cacgaggacc cagaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaacgcc 1020
aagacaaagc ctagggagga gcagtacaac agcacataca gagtggtctc cgtgctgaca 1080
gtgctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtgtc caataaggcc 1140
ctgcctgccc ctatcgagaa gaccatcagc aaggccaagg gccagcctag agagcctcag 1200
gtttacacac tgcccccttc cagggacgag ctgacaaaga accaggtgtc cctgacctgc 1260
ctggtgaagg gattctaccc tagcgacatc gccgtggagt gggagtctaa tggccagcca 1320
gagaataact acaagacaac cccccccgtg ctggattccg atggatcttt ctttctgtac 1380
agcaagctga cagtggataa gtccaggtgg cagcagggca acgtgttttc ctgtagcgtg 1440
atgcacgagg ccctgcacaa tcactacacc cagaagagcc tgagcctgag ccctggaaag 1500
<210> 23
<211> 500
<212> PRT
<213> 人工合成
<400> 23
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Asp Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Ala Ala Glu Pro Lys Ser
260 265 270
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
275 280 285
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440 445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Pro Gly Lys
500
<210> 24
<211> 1500
<212> DNA
<213> 人工合成
<400> 24
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaatatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttaaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggaggccgc cgccgagccc aagtccagcg ataagacaca cacctgccct 840
ccttgtcccg cccctgaact gctgggagga ccttctgtgt tcctgttccc ccccaagcct 900
aaggacaccc tgatgatcag cagaaccccc gaggtgacct gtgtggtggt ggatgtgtcc 960
cacgaggacc cagaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaacgcc 1020
aagacaaagc ctagggagga gcagtacaac agcacataca gagtggtctc cgtgctgaca 1080
gtgctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtgtc caataaggcc 1140
ctgcctgccc ctatcgagaa gaccatcagc aaggccaagg gccagcctag agagcctcag 1200
gtttacacac tgcccccttc cagggacgag ctgacaaaga accaggtgtc cctgacctgc 1260
ctggtgaagg gattctaccc tagcgacatc gccgtggagt gggagtctaa tggccagcca 1320
gagaataact acaagacaac cccccccgtg ctggattccg atggatcttt ctttctgtac 1380
agcaagctga cagtggataa gtccaggtgg cagcagggca acgtgttttc ctgtagcgtg 1440
atgcacgagg ccctgcacaa tcactacacc cagaagagcc tgagcctgag ccctggaaag 1500
<210> 25
<211> 500
<212> PRT
<213> 人工合成
<400> 25
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ala Ala Ala Glu Pro Lys Ser
260 265 270
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
275 280 285
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440 445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Pro Gly Lys
500
<210> 26
<211> 825
<212> DNA
<213> 人工合成
<400> 26
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggagcacca ccaccaccat caccaccacc atcat 825
<210> 27
<211> 275
<212> PRT
<213> 人工合成
<400> 27
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu His His His His His His His
260 265 270
His His His
275
<210> 28
<211> 825
<212> DNA
<213> 人工合成
<400> 28
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaagatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttgaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgacgaca caacagacgc tgtgagagat 780
cctcagaccc tggagcacca ccaccaccat caccaccacc atcat 825
<210> 29
<211> 275
<212> PRT
<213> 人工合成
<400> 29
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Asp Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu His His His His His His His
260 265 270
His His His
275
<210> 30
<211> 825
<212> DNA
<213> 人工合成
<400> 30
agagtgcagc ccaccgagag catcgtgagg tttcccaaca tcacaaatct gtgccctttc 60
ggcgaggtgt tcaacgccac aaggttcgcc agcgtgtacg cctggaatag aaagagaatc 120
tccaattgcg tggccgatta ctccgtgctg tacaatagcg ccagcttttc cacattcaag 180
tgctacggcg tgtcccccac caagctgaac gacctgtgct ttaccaatgt gtacgccgat 240
agcttcgtga tcaggggcga cgaggtgaga cagatcgccc ctggacagac aggcaatatc 300
gccgattaca attacaagct gcctgatgac tttaccggct gcgtgatcgc ctggaactcc 360
aataacctgg acagcaaggt gggcggcaac tacaactacc tgtacaggct gttcagaaag 420
agcaatctga agccctttga gagggacatc agcaccgaga tctaccaggc cggcagcaca 480
ccatgcaatg gcgttaaggg cttcaactgt tacttccccc tgcagtccta cggctttcag 540
cccacatacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt tgagctgctg 600
cacgcccctg ctaccgtgtg tggacctaag aagtccacca acctggtgaa gaataagtgc 660
gtgaacttta acttcaacgg cctgacaggc acaggcgtgc tgacagaatc caacaagaag 720
tttctgcctt tccagcagtt cggcagggac atcgccgaca caacagacgc tgtgagagat 780
cctcagaccc tggagcacca ccaccaccat caccaccacc atcat 825
<210> 31
<211> 275
<212> PRT
<213> 人工合成
<400> 31
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu His His His His His His His
260 265 270
His His His
275
<210> 32
<211> 696
<212> DNA
<213> 人工合成
<400> 32
gagcccaagt ccagcgataa gacacacacc tgccctcctt gtcccgcccc tgaactgctg 60
ggaggacctt ctgtgttcct gttccccccc aagcctaagg acaccctgat gatcagcaga 120
acccccgagg tgacctgtgt ggtggtggat gtgtcccacg aggacccaga ggtgaagttc 180
aactggtacg tggatggcgt ggaggtgcac aacgccaaga caaagcctag ggaggagcag 240
tacaacagca catacagagt ggtctccgtg ctgacagtgc tgcaccagga ctggctgaac 300
ggcaaggagt acaagtgcaa ggtgtccaat aaggccctgc ctgcccctat cgagaagacc 360
atcagcaagg ccaagggcca gcctagagag cctcaggttt acacactgcc cccttccagg 420
gacgagctga caaagaacca ggtgtccctg acctgcctgg tgaagggatt ctaccctagc 480
gacatcgccg tggagtggga gtctaatggc cagccagaga ataactacaa gacaaccccc 540
cccgtgctgg attccgatgg atctttcttt ctgtacagca agctgacagt ggataagtcc 600
aggtggcagc agggcaacgt gttttcctgt agcgtgatgc acgaggccct gcacaatcac 660
tacacccaga agagcctgag cctgagccct ggaaag 696
<210> 33
<211> 232
<212> PRT
<213> 人工合成
<400> 33
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 34
<211> 2769
<212> DNA
<213> 人工合成
<400> 34
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgtgtgaa tctgacaacc 60
agaacccagc tgcctcctgc ctacaccaac agctttacaa gaggcgtgta ctaccctgat 120
aaggtgttca ggagcagcgt gctgcactcc acccaggacc tgtttctgcc tttctttagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacaa acggcaccaa gaggttcgac 240
aatcctgtgc tgccttttaa cgacggcgtg tactttgcca gcacagagaa gagcaatatc 300
atcaggggct ggatctttgg caccaccctg gactccaaga cacagagcct gctgatcgtg 360
aacaacgcca caaacgtggt cattaaggtg tgtgagtttc agttttgcaa tgaccccttc 420
ctgggcgtgt actatcacaa gaacaataag tcctggatgg agagcgagtt tagggtgtac 480
agcagcgcca acaactgtac attcgagtac gtgtcccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaattttaa gaacctgagg gagtttgtgt ttaagaacat cgacggctac 600
tttaagatct acagcaagca cacacccatc aatctggtga gggatctgcc tcagggcttt 660
agcgccctgg agcccctggt ggatctgccc atcggcatca acatcacaag gtttcagaca 720
ctgctggccc tgcacagaag ctacctgacc cctggcgata gcagctccgg ctggaccgcc 780
ggagccgctg cttactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaacggca caatcaccga tgccgtggac tgcgccctgg atcccctgag cgagacaaag 900
tgtacactga agagcttcac cgtggagaag ggcatctacc agaccagcaa ctttagagtg 960
cagcccacag agagcatcgt gagattccct aacatcacca atctgtgtcc ctttggcgag 1020
gtgttcaatg ccacaaggtt tgcctccgtg tacgcctgga ataggaagag gatcagcaac 1080
tgtgtggccg attactccgt gctgtacaac agcgcctcct ttagcacctt taagtgttac 1140
ggcgtgtccc ccaccaagct gaacgatctg tgtttcacaa atgtgtacgc cgatagcttt 1200
gtgatcaggg gcgacgaggt gagacagatc gcccctggcc agaccggcaa gatcgccgac 1260
tacaattaca agctgcctga tgacttcacc ggctgtgtga tcgcctggaa tagcaataat 1320
ctggactcca aagtgggcgg caactacaac tacctgtaca gactgtttag aaagagcaac 1380
ctgaagccct tcgagaggga tatcagcacc gagatctacc aggccggctc cacaccttgc 1440
aatggcgtgg agggctttaa ctgttacttc cctctgcagt cctacggctt ccagcccaca 1500
aatggcgtgg gctaccagcc ttacagggtg gtggtgctga gctttgagct gctgcacgcc 1560
cctgccacag tgtgtggccc taagaagagc accaacctgg tgaagaataa gtgcgtgaac 1620
ttcaatttca atggcctgac cggcaccggc gtgctgaccg aaagcaacaa gaagtttctg 1680
cccttccagc agtttggcag agacatcgcc gacaccaccg acgccgtgag ggatccccag 1740
accctggaga tcctggatat caccccttgc tcctttggcg gcgtgtccgt gatcacccct 1800
ggcacaaaca catccaacca ggtggccgtg ctgtaccagg atgtgaactg caccgaggtg 1860
cctgtggcca tccacgccga tcagctgacc cctacctgga gggtgtactc caccggctcc 1920
aatgtgtttc agaccagagc cggctgtctg atcggcgccg agcacgtgaa caattcctac 1980
gagtgcgaca tccccatcgg cgccggcatc tgcgcctcct accagaccca gacaaacagc 2040
cctaggagag ccagagccga cgacgatgac aaggagccca agtccagcga taagacacac 2100
acctgccctc cttgtcccgc ccctgaactg ctgggaggac cttctgtgtt cctgttcccc 2160
cccaagccta aggacaccct gatgatcagc agaacccccg aggtgacctg tgtggtggtg 2220
gatgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggatgg cgtggaggtg 2280
cacaacgcca agacaaagcc tagggaggag cagtacaaca gcacatacag agtggtctcc 2340
gtgctgacag tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 2400
aataaggccc tgcctgcccc tatcgagaag accatcagca aggccaaggg ccagcctaga 2460
gagcctcagg tttacacact gcccccttcc agggacgagc tgacaaagaa ccaggtgtcc 2520
ctgacctgcc tggtgaaggg attctaccct agcgacatcg ccgtggagtg ggagtctaat 2580
ggccagccag agaataacta caagacaacc ccccccgtgc tggattccga tggatctttc 2640
tttctgtaca gcaagctgac agtggataag tccaggtggc agcagggcaa cgtgttttcc 2700
tgtagcgtga tgcacgaggc cctgcacaat cactacaccc agaagagcct gagcctgagc 2760
cctggaaag 2769
<210> 35
<211> 923
<212> PRT
<213> 人工合成
<400> 35
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Asp Asp
675 680 685
Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
690 695 700
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
705 710 715 720
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
725 730 735
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
740 745 750
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
755 760 765
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
770 775 780
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
785 790 795 800
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
805 810 815
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
820 825 830
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
835 840 845
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
850 855 860
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
865 870 875 880
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
885 890 895
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
900 905 910
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
915 920
<210> 36
<211> 3672
<212> DNA
<213> 人工合成
<400> 36
atgttcgtgt ttctggtgct gctgcccctg gtgagctccc aatgtgtgaa tctgaccaca 60
aggacacagc tgcctcctgc ctacaccaac agctttacca ggggcgtgta ctaccctgac 120
aaggtgttca gaagctccgt gctgcactcc acccaggacc tgttcctgcc tttctttagc 180
aatgtgacat ggtttcacgc catccacgtg tccggcacca acggaacaaa gaggtttgat 240
aatcccgtgc tgcccttcaa cgatggcgtg tactttgcct ccaccgagaa gtccaacatc 300
atcagaggct ggatctttgg cacaacactg gacagcaaga cacagagcct gctgatcgtg 360
aacaacgcca caaatgtggt cattaaggtg tgtgagttcc agttctgcaa cgatcccttc 420
ctgggcgtgt actatcacaa gaataacaag agctggatgg agtccgagtt cagggtgtac 480
agcagcgcca acaattgcac atttgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaactttaa gaacctgaga gagttcgtgt tcaagaacat cgatggctac 600
ttcaagatct acagcaagca cacacccatc aacctggtga gggatctgcc tcagggcttt 660
tccgccctgg agcctctggt tgacctgcct atcggcatca atatcaccag attccagacc 720
ctgctggccc tgcacaggtc ttacctgacc cctggagatt ccagctccgg ctggacagct 780
ggagctgctg cttactacgt gggctacctg cagcccagaa ccttcctgct gaagtacaac 840
gagaatggca caatcaccga cgccgtggac tgtgctctgg accctctgtc tgagacaaag 900
tgtaccctga agagcttcac agtggagaag ggcatctacc agaccagcaa tttcagagtg 960
cagcccaccg agtccatcgt gagatttccc aatatcacca acctgtgtcc ctttggcgag 1020
gtgttcaacg ccacaagatt cgccagcgtg tacgcctgga acaggaagag aatcagcaac 1080
tgcgtggccg actactccgt gctgtacaat agcgccagct ttagcacctt caagtgctac 1140
ggcgtgagcc ctaccaagct gaacgacctg tgtttcacca acgtgtacgc cgatagcttt 1200
gtgatcaggg gcgatgaggt gaggcagatc gcccctggac agacaggaaa gatcgccgac 1260
tacaactaca agctgcctga tgactttacc ggctgtgtga tcgcctggaa ctccaataat 1320
ctggatagca aggtgggcgg caattacaat tacctgtaca gactgtttag aaagagcaat 1380
ctgaagccct ttgagaggga tatctccaca gagatctacc aggccggctc caccccatgt 1440
aatggcgttg agggctttaa ttgctacttt cctctgcaga gctacggctt tcagcccacc 1500
aacggcgtgg gataccagcc ttacagggtg gtggtgctga gcttcgagct gctgcacgct 1560
cctgctacag tgtgcggacc taagaagagc acaaacctgg tgaagaataa gtgcgtgaat 1620
tttaacttca acggcctgac cggcaccggc gttctgacag aaagcaataa gaagttcctg 1680
ccttttcagc agtttggcag agacatcgcc gacaccacag atgccgtgag ggaccctcag 1740
accctggaaa tcctggacat cacaccctgc agctttggcg gcgtgtccgt tatcaccccc 1800
ggaacaaata ccagcaatca ggtggccgtg ctgtaccagg acgtgaactg cacagaggtg 1860
cccgtggcta tccacgctga tcagctgaca cccacatgga gagtgtactc caccggctcc 1920
aacgtgttcc agacaagagc cggctgtctg atcggcgctg agcatgttaa caattcctac 1980
gagtgcgaca tccccatcgg cgctggaatc tgtgccagct accagaccca gaccaacagc 2040
cctaggagag ccagatccgt ggcctctcag agcatcatcg cctacacaat gtccctgggc 2100
gccgagaact ccgtggctta ttccaataac agcatcgcca tccccaccaa ctttaccatc 2160
agcgtgacca cagagatcct gcccgtgtcc atgaccaaga caagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgtagc aatctgctgc tgcagtacgg cagcttctgt 2280
acccagctga atagagccct gacaggcatc gccgtggagc aagataagaa tacacaggag 2340
gtgtttgccc aggtgaagca gatctacaag acccctccta tcaaggactt cggcggcttt 2400
aactttagcc agatcctgcc tgatcccagc aagcctagca agaggagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta cggcgactgt 2520
ctgggcgata tcgccgctag agacctgatc tgcgcccaga agttcaacgg cctcacagtg 2580
ctgcctcctc tgctgacaga tgagatgatc gcccagtaca catccgccct gctggctgga 2640
accatcacaa gcggatggac attcggcgcc ggcgctgctc tgcaaatccc ttttgccatg 2700
cagatggcct acaggtttaa tggcatcggc gtgacacaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt caatagcgcc atcggcaaga tccaggacag cctgagctcc 2820
acagccagcg ctctgggaaa gctgcaggac gttgtgaatc agaatgccca ggccctgaat 2880
acactggtga agcagctgag cagcaacttt ggcgccatca gcagcgtgct gaatgatatc 2940
ctgtccagac tggataaggt ggaggccgag gtgcagatcg ataggctgat caccggcaga 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatca gggccgctga aatcagggcc 3060
agcgctaatc tggccgccac aaagatgagc gagtgtgtgc tgggccagag caagagagtg 3120
gacttttgtg gcaagggcta ccacctgatg tccttccctc agagcgcccc acacggagtt 3180
gtgttcctgc acgtgacata cgtgcccgcc caggaaaaga actttaccac cgcccctgcc 3240
atctgccacg atggaaaggc tcacttcccc agagagggcg tgtttgtgag caatggcacc 3300
cactggttcg tgacccagag gaacttttac gagcctcaga tcatcaccac agataacaca 3360
tttgtgagcg gcaactgtga cgtggtcatt ggcatcgtga ataacacagt gtacgacccc 3420
ctgcagcccg agctggattc tttcaaggag gagctggaca agtacttcaa gaaccacaca 3480
agccccgatg tggacctggg cgatattagc ggcatcaatg ccagcgtggt gaacatccag 3540
aaggagatcg atagactgaa cgaggtggcc aagaatctga acgagagcct gatcgacctg 3600
caggagctgg gaaagtacga gcagtacatc aagtggcccg cccaccacca ccaccatcac 3660
catcaccacc ac 3672
<210> 37
<211> 1224
<212> PRT
<213> 人工合成
<400> 37
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Ala His His His His
1205 1210 1215
His His His His His His
1220

Claims (13)

1.一种融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID No.21、SEQ IDNo.23、SEQ ID No.25任一所述。
2.编码权利要求1所述融合蛋白的核酸分子。
3.根据权利要求2所述的核酸分子，其特征在于，所述核酸分子的序列是指SEQ IDNo.20、SEQ ID No.22、SEQ ID No.24任一所示的多核苷酸序列或其简并序列。
4.包含权利要求2或3所述核酸分子的载体。
5.根据权利要求4所述的载体，其特征在于，所述载体是将所述核酸分子连接到质粒pcDNA3.1中得到。
6.包含权利要求2或3所述核酸分子或权利要求4~5任一所述载体的细胞。
7.根据权利要求6所述的细胞，其特征在于，所述细胞为将核酸分子或载体转入宿主细胞Expi293F中得到。
8.一种疫苗组合物，其特征在于，所述疫苗组合物包含权利要求1所述的融合蛋白，权利要求2或3所述核酸分子，或权利要求4或5所述的载体。
9.一种多价疫苗组合物，其特征在于，所述多价疫苗组合物包含至少两种如权利要求1中所述融合蛋白。
10.根据权利要求8所述的疫苗组合物或权利要求9所述的多价疫苗组合物，其特征在于，所述疫苗组合物或多价疫苗组合物还包括免疫学和药学上接受的载体或佐剂。
11.根据权利要求10任一所述的疫苗组合物或多价疫苗组合物，其特征在于，所述佐剂包括铝佐剂、弗氏佐剂、磷酸钙、石蜡油、羊毛脂、藻酸钙、胞壁肽、皂苷、RIBI佐剂系统、霍乱毒素、丙烯酸或甲基丙烯酸的聚合物中的一种或几种。
12.根据权利要求11任一所述的疫苗组合物或多价疫苗组合物，其特征在于，所述佐剂为铝佐剂。
13.根据权利要求10任一所述疫苗组合物或多价疫苗组合物在制备预防和/或治疗新型冠状病毒肺炎药物中的应用。
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