CN114349870B - 一种兼具防粘连功效的重组抗菌肽及其制备方法和应用 - Google Patents

一种兼具防粘连功效的重组抗菌肽及其制备方法和应用 Download PDF

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CN114349870B
CN114349870B CN202210274204.9A CN202210274204A CN114349870B CN 114349870 B CN114349870 B CN 114349870B CN 202210274204 A CN202210274204 A CN 202210274204A CN 114349870 B CN114349870 B CN 114349870B
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许洋
李慧
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Sensor Control Eagle Fly (Tianjin) Medical Technology Co.,Ltd.
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Abstract

本发明提供了一种兼具防粘连功效的重组抗菌肽及其制备方法和应用，所述重组抗菌肽由氨基酸序列如SEQ ID NO.1所示的抗菌肽与如下蛋白片段a或片段b连接而成；片段a：氨基酸序列如SEQ ID NO.2所示；片段b：氨基酸序列如SEQ ID NO.3所示。本发明是通过添加或者促进纤溶酶的活性，从而维持纤溶酶与凝血酶之间的平衡，达到预防术后粘连症状的发生。同时防粘连蛋白与抗菌肽进行融合得到的重组抗菌肽既可以预防术后粘连症状的发生，又能防止术后伤口被细菌感染。

Description

一种兼具防粘连功效的重组抗菌肽及其制备方法和应用
技术领域
本发明创造属于化妆品及医学美容技术领域，尤其是涉及一种兼具防粘连功效的重组抗菌肽及其制备方法和应用。
背景技术
肠黏连是盆腹腔术后常见并发症之一，其发生率高达90%以上，其中60%-70%的患者可进展为肠梗阻；此外，肠黏连还可引发慢性腹痛及不孕症，并大大增加腹腔再次手术难度。肠黏连是腹腔受损失、感染刺激后的防御反应，多为纤维蛋白沉淀于腹膜表面未能完全吸收所致。如何防治术后肠黏连是临床外科医师面临的严峻挑战。
目前市场上多采用腹腔内用药以降低术后肠黏连的发生率。采用粘停宁联合糜蛋白预防盆腔黏连。氧化再生纤维素是经过氧化氮处理可吸收人造纤维补片，能转为凝胶状物质覆盖在受损腹膜表面而有效减少粘连发生。生物可吸收防粘连膜是以透明质酸一羧甲基纤维素为主要成分，可为损伤组织提供长达7d的保护膜。可降解抗组织粘连膜是由乳酸共聚物组成的可吸收透明膜，能有效减少术后粘连。膨化聚四氟乙烯是具有双面结构的不可吸收薄膜，必须缝合在组织上，可有效预防粘连的形成。也有使用一些干扰炎症反应来减少粘连形成的药物，能减轻炎症反应的皮质类固醇、抗组胺药、非甾体类抗炎药以及环氧化酶2抑制剂。但这些药物的免疫抑制和伤口延迟愈合的潜在不良反应限制了其临床应用。对于不可吸收的机械屏障由于其疏水性、组织粘附性差及非生物降解性，大多数医师担心其会增加缝合部位感染的机会而不愿使用。
综上所示，目前市场上多采用可降解的防黏连膜，防黏连作用的腹腔冲洗液，透明质酸一羧甲基纤维素。虽然这些防黏连的膜和冲洗液对术后的黏连症状有一定的预防作用，但是这些膜和冲洗液对身体的自身代谢有很大的干扰作用，且不能从根本上来解决术后黏连症状的发生，同时又不能预防细菌对伤口的感染。
因此开发一种既能预防术后粘连症状，又能防止鲍曼不动杆菌的感染以满足预防术后粘连症状的发生在生物医学领域的应用显得十分重要。
发明内容
有鉴于此，本发明针对这些存在的问题，利用防黏连蛋白和抗菌肽蛋白进行融合表达，获得既能对术后伤口发生的黏连症状进行预防，又能防止细菌对术后伤口的感染，这在生物医药领域具有重要的应用价值。
为达到上述目的，本发明创造的技术方案是这样实现的：
一种兼具防粘连功效的重组抗菌肽，所述重组抗菌肽由氨基酸序列如SEQ IDNO.1所示的抗菌肽与如下蛋白片段a或片段b连接而成；
片段a：氨基酸序列如SEQ ID NO.2所示；
片段b：氨基酸序列如SEQ ID NO.3所示。
优选的，重组抗菌肽由抗菌肽与蛋白片段a或片段b通过连接肽连接而成，连接肽的氨基酸序列如SEQ ID NO.4所示，核苷酸序列如SEQ ID NO.5所示。
优选的，所述重组抗菌肽的氨基酸序列如SEQ ID NO.6或SEQ ID NO.7所示,。
本发明还提供了一种编码上述重组抗菌肽的基因。
优选的，所述基因包括如SEQ ID NO.8所示的编码抗菌肽的核苷酸序列和如下编码蛋白片段a的基因或编码蛋白片段b的基因连接而成：
编码蛋白片段a的核苷酸序列如SEQ ID NO.9所示；
编码蛋白片段b的核苷酸序列如SEQ ID NO.10所示。
优选的，编码所述重组抗菌肽的核苷酸序列如SEQ ID NO.11或SEQ ID NO.12所示。
本发明还提供一种含有编码上述重组抗菌肽的核苷酸序列的表达载体。
优选的，所述表达载体为Pet32A质粒。
本发明还提供一种含有编码上述重组抗菌肽的核苷酸序列的宿主细胞。
优选的，所述宿主细胞为大肠杆菌BL21。
本发明还提供一种上述重组抗菌肽在制备预防术后粘连及术后伤口被细菌感染药物中的应用。
相对于现有技术，本发明具有以下优势：
本发明所述的重组抗菌肽是利用组织纤溶酶原激活剂和组织纤溶酶原来预防术后伤口发生粘连症状。粘连症状的发生原理是由于：腹膜损伤后，受损区域首先被炎症细胞浸润，趋化信号由沉积的血小板释放，同时生成前列腺素类和白三烯类物质，间皮细胞被募集到损伤位点。随后富含纤维的间质在损伤部位表达，创面间形成纤维间质桥接，如果纤维蛋白溶解活性足够强，将溶解纤维性粘连，否则形成结缔组织。粘连进一步发展。炎性细胞的聚集、间皮细胞的活化、纤维素的渗出和细胞外基质的转化与重塑均在粘连的形成过程中起作用。但粘连的形成主要取决于凝血酶和纤溶酶之间的平衡。纤溶酶的活性主要由组织型纤溶酶原激活物(t-PA)与纤溶酶原激活物抑制剂1(PAI．1)的相对水平决定，而在盆腔手术后腹膜的再覆过程中，t-PA和PAI．1的平衡常偏向于粘连形成和发展。本发明是通过添加或者促进纤溶酶的活性，从而维持纤溶酶与凝血酶之间的平衡，达到预防术后粘连症状的发生。同时防粘连蛋白与抗菌肽进行融合得到的重组抗菌肽既可以预防术后粘连症状的发生，又能防止术后伤口被细菌感染。
附图说明
图1为重组质粒Pet32a-A3的构建图谱；
图2为重组质粒Pet32a -plat-A3的构建图谱；
图3为重组质粒Pet32a- plg -A3的构建图谱。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的实验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明创造。
一、抗菌肽的设计
从天然的抗菌肽中，筛选出高活性的抗菌肽。将抗菌肽进行剪切，将剪切后的片段进行拼接，同时对拼接后的抗菌肽进行突变，以获得更高活性的抗菌肽。得到的抗菌肽的氨基酸序列为：
1、螺旋抗菌肽的设计
第一条：
GLKEIFKAGLGSLVKGIAAHVASAGPGIGKFLHSAGKFGKAFVGEIMKS；
第二条：
GLKEIFKAGLGSLVKGIAAHVASAGPSAVHAAIGKVLSGLGAKFIEKLG；
第三条：
SKMIEGVFAKGLKFASHLFKGIGAGPGIGKFLHSAGKFGKAFVGEIMKS；
2、线性抗菌肽的设计
第一条：FRFWFRFRFGSGFRFRFWFRF；
第二条：FIFFPFTSLKSFFISSPFFSPRRYKQIGTFGLPGTSFFSKP；
第三条：SFVFRFWFRFFLRKFRKF；
第四条：SFVFRFWFRFFLRKFRKFGSGFKRFKRLFFRFWFRFVFS。
3、抗菌肽碱基密码子序列的优化
3.1、螺旋抗菌肽密码子序列的优化
第一条序列A1：（EcoRI/HindIII）
GAATTCGGCCTGAAAGAAATCTTCAAAGCCGGTCTGGGTAGCCTGGTGAAAGGTATCGCTGCGCACGTAGCTTCTGCTGGTCCGGGTATCGGTAAATTTCTGCATTCCGCTGGCAAATTTGGCAAAGCATTCGTCGGTGAAATCATGAAAAGCTAAAAGCTT
第二条序列A2：（EcoRI/HindIII）
GAATTCGGTCTGAAAGAAATCTTCAAAGCTGGTCTGGGTTCTCTGGTTAAAGGTATCGCTGCTCACGTTGCTTCTGCTGGTCCGTCTGCTGTTCACGCTGCTATCGGTAAAGTTCTGTCTGGTCTGGGTGCTAAATTCATCGAAAAACTGGGTTAAAAGCTT
第三条序列A3：（EcoRI/HindIII）
GAATTCAGCAAAATGATCGAAGGTGTTTTTGCGAAAGGTCTGAAATTTGCATCTCACCTGTTCAAAGGCATCGGTGCAGGTCCGGGTATCGGTAAATTCCTGCATTCTGCGGGCAAATTTGGCAAAGCGTTCGTCGGCGAAATCATGAAATCTTAAAAGCTT
3.2、线性抗菌肽密码子序列的优化
第一条序列X1：（EcoRI/HindIII）
GAATTCTTCCGTTTCTGGTTCCGTTTCCGTTTCGGTTCTGGTTTCCGTTTCCGTTTCTGGTTCCGTTTCTAAAAGCTT
第二条序列X2：（EcoRI/HindIII）
GAATTCTTCATCTTCTTCCCGTTCACCTCTCTGAAATCTTTCTTCATCTCTTCTCCGTTCTTCTCTCCGCGTCGTTACAAACAGATCGGTACCTTCGGTCTGCCGGGTACCTCTTTCTTCTCTAAACCGTAAAAGCTT
第三条序列X3：（EcoRI/HindIII）
GAATTCTCTTTCGTTTTCCGTTTCTGGTTCCGTTTCTTCCTGCGTAAATTCCGTAAATTCTAAAAGCTT
第四条序列X4：（EcoRI/HindIII）
GAATTCTCTTTCGTTTTCCGTTTCTGGTTCCGTTTCTTCCTGCGTAAATTCCGTAAATTCGGTTCTGGTTTCAAACGTTTCAAACGTCTGTTCTTCCGTTTCTGGTTCCGTTTCGTTTTCTCTTAAAAGCTT
4、抗菌肽酶切位点的选择
选择大肠杆菌BL21为宿主，以质粒Pet32a为载体，进行重组质粒的构建。通过SNAPGENE分析出载体和抗菌肽的酶切位点。选择合适的酶切位点进行酶切连接，A1（EcoRI/HindIII）、A2（EcoRI/HindIII）、A3（EcoRI/HindIII）、X1（EcoRI/HindIII）、X2（EcoRI/HindIII）、X3（EcoRI/HindIII）、X4（EcoRI/HindIII），如图1所示。将所切获得的相同黏性末端的片段，在连接酶的作用，进行连接。
二、重组菌种的构建
A1、A2、A3、X1、X2、X3、X4序列均由金唯智公司合成，且直接构建到质粒Pet32a上，形成重组大肠杆菌BL21/Pet32a-A1、BL21/Pet32a-A2、BL21/Pet32a-A3、BL21/Pet32a-X1、BL21/Pet32a-X2、BL21/Pet32a-X3、BL21/Pet32a-X4。
三、抗菌肽的获得
挑取重组大肠杆菌单菌落接种到5 mL LB液体培养基中，37℃、220 r/min培养过夜，将菌液接种到50 mL LB发酵培养基中，控制起始菌浓OD600=0.1，待菌株的OD600=0.6时，加入适量的诱导剂IPTG，使其诱导剂的终浓度为0.5 mmoL/L，18℃、180 r/min 诱导24h后。收集菌体，破碎细胞，离心得上清，将上清通过Ni-柱纯化，得到较纯的抗菌肽。将获得的抗菌肽，进行肠激酶酶切。酶切后的溶液进行Ni-柱纯化，得到抗菌肽产物。
四、抗菌肽的活性检测分析
将获得的抗菌肽产物，进行抑菌活性检测。选择大肠杆菌、鲍曼不动杆菌、金黄色葡萄球菌来检测抗菌肽的抑菌效果。将三种菌活化后，涂布在LB平板上，在LB平板上放置滤纸，且在滤纸上滴加含有适量的抗菌肽溶液。将LB平板在37℃条件下培养48小时，观察实验组与空白对照组的细菌生长情况（空白对照组：用生理盐水代替抗菌肽溶液）。试验结果见下表：
表1 抗菌肽产物的抑菌活性检测结果
Figure DEST_PATH_IMAGE001
从表1的数据结果可以看出，抗菌肽A3的抗菌效果明显优于其他抗菌肽。
五、重组抗菌肽的构建
选择组织纤溶蛋白酶和组织纤溶蛋白酶激活剂作为防粘连蛋白，均由金唯智公司合成，且直接插入到Pet32a-A3的质粒中，形成重组大肠杆菌BL21/ Pet32A -plg-A3、BL21/Pet32A-plat-A3。
六、重组抗菌肽的制备
挑取重组大肠杆菌单菌落接种到5 mL LB液体培养基中，37℃、220 r/min培养过夜，将菌液接种到50 mL LB发酵培养基中，控制起始菌浓OD600=0.1，待菌株的OD600=0.6时，加入适量的诱导剂IPTG，使其诱导剂的终浓度为0.5 mmoL/L，18℃、180 r/min 诱导24h后。收集菌体，破碎细胞，离心得上清，将上清通过Ni-柱纯化，得到较纯的重组抗菌肽。将获得的重组抗菌肽，进行肠激酶酶切。酶切后的溶液进行Ni-柱纯化，得到重组抗菌肽产物。
七、重组抗菌肽的活性检测分析
肠粘连模型制备：所有动物禁食不禁水12 h， 以30 g/L戊巴比妥钠溶液按30 mg/kg肌肉注射 (避免腹腔注射可能引起的肠粘连)麻醉。取下腹部正中切口约2 cm，取出盲肠，轻刮盲肠浆膜表面，范围为Φ3 cm左右区域，直至渗血，再滴无水乙醇于创面上，然后以五齿镊夹住盲肠系膜动脉约2 min，造成暂时局部缺血。经上述处理后，纤溶酶原激活剂组、纤溶酶原组、纤溶酶原激活剂重组抗菌肽组和纤溶酶原重组抗菌肽组分别以相同剂量(1g)药物完全覆盖创面；对照组不给予任何药物。用药后回纳盲肠入腹腔原位后以止血钳夹伤相应腹壁，分层以1-0号丝线关闭腹腔。术后每天肌注庆大霉素4U，连续3d，预防感染。14d后以同样的麻醉方法开腹检验、取材。主要观察指标：大体观察：观察动物伤口和腹腔感染情况，比较各组药物体内残留情况。粘连程度判断：术后14 d，全部动物剖腹，为能更好地观察腹腔粘连情况，再次腹腔切口时包含原正中切口在内的、以底朝下的“U”口切开腹腔，然后将腹壁组 织瓣向上掀起暴露腹腔，观察盲肠末端与腹壁创伤处之间的粘连情况。粘连程度采用半定量的分级评定法，参照Nair等制定的分级标准：0级，完全无粘连；1级，内脏间或腹壁间1条粘连带；2级，内脏间或内脏与腹壁间2条粘连带；3级，多于2条粘连带，而内脏未直接粘连到腹壁；4级，内脏直接粘连到腹壁，而不管粘连带多少。
表2 肠粘连程度分级结果
（各组大鼠肠粘连程度，各组大鼠肠粘连评级结果 (n=10)）
Figure 10372DEST_PATH_IMAGE002
从表2的数据结果可以看出，纤溶酶原激活剂重组抗菌肽实验组和纤溶酶原重组抗菌肽实验组的粘连程度明显低于对照组，且重组抗菌肽比纤溶酶原激活剂和纤溶酶原均低0.2和0.1，出现协同增效作用，表明重组抗菌肽对术后的粘连具有较好的预防效果。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
序列表
<110> 天津辅元生物医药科技有限公司
<120> 一种兼具防粘连功效的重组抗菌肽及其制备方法和应用
<141> 2022-01-21
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 51
<212> PRT
<213> Artificial Sequence
<400> 1
Glu Phe Ser Lys Met Ile Glu Gly Val Phe Ala Lys Gly Leu Lys Phe
1 5 10 15
Ala Ser His Leu Phe Lys Gly Ile Gly Ala Gly Pro Gly Ile Gly Lys
20 25 30
Phe Leu His Ser Ala Gly Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
35 40 45
Met Lys Ser
50
<210> 2
<211> 527
<212> PRT
<213> Artificial Sequence
<400> 2
Ser Tyr Gln Val Ile Cys Arg Asp Glu Lys Thr Gln Met Ile Tyr Gln
1 5 10 15
Gln His Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu
20 25 30
Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser Val Pro Val
35 40 45
Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys Gln Gln
50 55 60
Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu Gly Phe Ala
65 70 75 80
Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gln
85 90 95
Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Gly Ala Glu
100 105 110
Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys Pro Tyr Ser Gly
115 120 125
Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His Asn Tyr Cys
130 135 140
Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala
145 150 155 160
Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly
165 170 175
Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His
180 185 190
Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile
195 200 205
Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu
210 215 220
Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys
225 230 235 240
Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys
245 250 255
Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro
260 265 270
Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro
275 280 285
Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg
290 295 300
Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala
305 310 315 320
Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Ile
325 330 335
Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe
340 345 350
Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr
355 360 365
Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys
370 375 380
Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp
385 390 395 400
Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys
405 410 415
His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His
420 425 430
Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn
435 440 445
Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly
450 455 460
Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly
465 470 475 480
Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile
485 490 495
Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr
500 505 510
Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro
515 520 525
<210> 3
<211> 791
<212> PRT
<213> Artificial Sequence
<400> 3
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
1 5 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu
145 150 155 160
Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly
165 170 175
Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 185 190
Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys
195 200 205
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro
210 215 220
Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile
225 230 235 240
Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys
245 250 255
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val
260 265 270
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His
275 280 285
Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr
290 295 300
Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn
305 310 315 320
Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser
325 330 335
Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr
340 345 350
Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly
355 360 365
Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser
370 375 380
Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala
385 390 395 400
Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro
405 410 415
Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430
Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val
435 440 445
Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe
450 455 460
Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly
465 470 475 480
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile
485 490 495
Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys
500 505 510
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn
515 520 525
Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro
530 535 540
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
545 550 555 560
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
565 570 575
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
580 585 590
Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
595 600 605
Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val
610 615 620
Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu
625 630 635 640
Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala
645 650 655
Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr
660 665 670
Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr
675 680 685
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
690 695 700
Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
705 710 715 720
Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
725 730 735
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys
740 745 750
Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro
755 760 765
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile
770 775 780
Glu Gly Val Met Arg Asn Asn
785 790
<210> 4
<211> 12
<212> PRT
<213> Artificial Sequence
<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
1 5 10
<210> 5
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 5
ggtggtggtg gttctggtgg tggtggttct ggcggt 36
<210> 6
<211> 590
<212> PRT
<213> Artificial Sequence
<400> 6
Ser Tyr Gln Val Ile Cys Arg Asp Glu Lys Thr Gln Met Ile Tyr Gln
1 5 10 15
Gln His Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu
20 25 30
Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser Val Pro Val
35 40 45
Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys Gln Gln
50 55 60
Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu Gly Phe Ala
65 70 75 80
Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gln
85 90 95
Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Gly Ala Glu
100 105 110
Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys Pro Tyr Ser Gly
115 120 125
Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His Asn Tyr Cys
130 135 140
Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala
145 150 155 160
Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly
165 170 175
Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His
180 185 190
Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile
195 200 205
Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu
210 215 220
Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys
225 230 235 240
Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys
245 250 255
Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro
260 265 270
Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro
275 280 285
Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg
290 295 300
Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala
305 310 315 320
Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Ile
325 330 335
Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe
340 345 350
Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr
355 360 365
Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys
370 375 380
Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp
385 390 395 400
Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys
405 410 415
His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His
420 425 430
Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn
435 440 445
Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly
450 455 460
Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly
465 470 475 480
Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile
485 490 495
Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr
500 505 510
Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro Gly
515 520 525
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Phe Ser Lys Met
530 535 540
Ile Glu Gly Val Phe Ala Lys Gly Leu Lys Phe Ala Ser His Leu Phe
545 550 555 560
Lys Gly Ile Gly Ala Gly Pro Gly Ile Gly Lys Phe Leu His Ser Ala
565 570 575
Gly Lys Phe Gly Lys Ala Phe Val Gly Glu Ile Met Lys Ser
580 585 590
<210> 7
<211> 854
<212> PRT
<213> Artificial Sequence
<400> 7
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
1 5 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu
145 150 155 160
Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly
165 170 175
Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 185 190
Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys
195 200 205
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro
210 215 220
Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile
225 230 235 240
Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys
245 250 255
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val
260 265 270
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His
275 280 285
Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr
290 295 300
Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn
305 310 315 320
Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser
325 330 335
Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr
340 345 350
Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly
355 360 365
Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser
370 375 380
Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala
385 390 395 400
Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro
405 410 415
Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430
Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val
435 440 445
Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe
450 455 460
Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly
465 470 475 480
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile
485 490 495
Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys
500 505 510
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn
515 520 525
Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro
530 535 540
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
545 550 555 560
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
565 570 575
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
580 585 590
Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
595 600 605
Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val
610 615 620
Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu
625 630 635 640
Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala
645 650 655
Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr
660 665 670
Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr
675 680 685
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
690 695 700
Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
705 710 715 720
Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
725 730 735
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys
740 745 750
Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro
755 760 765
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile
770 775 780
Glu Gly Val Met Arg Asn Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly
785 790 795 800
Ser Gly Gly Glu Phe Ser Lys Met Ile Glu Gly Val Phe Ala Lys Gly
805 810 815
Leu Lys Phe Ala Ser His Leu Phe Lys Gly Ile Gly Ala Gly Pro Gly
820 825 830
Ile Gly Lys Phe Leu His Ser Ala Gly Lys Phe Gly Lys Ala Phe Val
835 840 845
Gly Glu Ile Met Lys Ser
850
<210> 8
<211> 162
<212> DNA
<213> Artificial Sequence
<400> 8
gaattcagca aaatgatcga aggtgttttt gcgaaaggtc tgaaatttgc atctcacctg 60
ttcaaaggca tcggtgcagg tccgggtatc ggtaaattcc tgcattctgc gggcaaattt 120
ggcaaagcgt tcgtcggcga aatcatgaaa tcttaaaagc tt 162
<210> 9
<211> 1629
<212> DNA
<213> Artificial Sequence
<400> 9
ggatcctcct accaggtaat ctgccgcgac gaaaaaaccc agatgatcta ccagcagcat 60
cagtcctggc tgcgtccggt tctgcgctct aaccgtgttg aatattgctg gtgcaactcc 120
ggtcgtgcgc aatgccattc tgttccggtg aaatcttgct ctgagccacg ttgttttaac 180
ggtggtacct gccagcaggc actgtatttt tccgacttcg tatgtcagtg cccggaaggt 240
tttgcgggta aatgctgtga aatcgatacc cgtgctactt gctatgagga ccagggtatc 300
agctatcgtg gtacttggtc caccgctgaa agcggtgcgg aatgtactaa ctggaacagc 360
tccgctctgg cgcagaaacc gtactctggt cgtcgtcctg atgccatccg tctgggtctg 420
ggcaaccata actactgccg taacccggat cgtgatagca aaccgtggtg ttacgtattt 480
aaagctggta agtactcttc cgagttctgc agcacgccgg cgtgtagcga aggcaacagc 540
gattgctact tcggcaacgg ttccgcctac cgtggtaccc actccctgac cgaatctggc 600
gcaagctgtc tgccatggaa ctctatgatc ctgatcggta aggtctacac cgctcagaac 660
ccgtctgcgc aggctctggg tctgggtaag cacaactact gtcgtaaccc ggatggtgat 720
gctaaaccat ggtgccatgt tctgaaaaac cgtcgtctga cctgggaata ctgcgacgtg 780
ccgtcttgct ccacgtgcgg tctgcgtcag tactcccagc cgcagttccg cattaaaggt 840
ggtctgttcg ctgatattgc ctctcacccg tggcaggcag ctattttcgc caaacatcgt 900
cgttctccgg gtgaacgctt cctgtgcggt ggcatcctga tcagctcttg ctggattctg 960
tctgccgctc actgttttca ggaacgtttc ccaccacacc atctgaccgt tatcctgggc 1020
cgtacctacc gtgttgttcc gggtgaggag gaacagaaat tcgaggtgga gaaatacatt 1080
gtgcataaag agttcgacga tgatacctac gataacgaca tcgcactgct gcagctgaaa 1140
tccgactcta gccgctgtgc acaggagtcc agcgtggttc gtaccgtttg cctgcctccg 1200
gctgacctgc aactgcctga ctggaccgaa tgcgaactgt ctggttacgg caaacacgag 1260
gcactgagcc cgttctactc tgaacgtctg aaagaagcgc acgtgcgtct gtatccttct 1320
agccgctgta ccagccagca cctgctgaac cgtaccgtga ctgacaacat gctgtgcgca 1380
ggcgatactc gttctggcgg tccacaggct aacctgcacg atgcgtgtca gggtgattct 1440
ggcggtccgc tggtatgtct gaacgacggt cgtatgactc tggttggcat catctcctgg 1500
ggtctgggtt gcggtcagaa agacgtacca ggtgtttata ccaaagttac caactacctg 1560
gattggattc gtgataacat gcgtcctggt ggtggtggtt ctggtggtgg tggttctggc 1620
ggtgaattc 1629
<210> 10
<211> 2421
<212> DNA
<213> Artificial Sequence
<400> 10
ggatccgaac cactggatga ctacgtaaat acgcagggtg cttccctgtt ctctgtaact 60
aaaaaacaac tgggcgctgg ttccattgaa gagtgcgcag ctaaatgtga agaggatgaa 120
gaatttacct gccgtgcttt ccagtaccac agcaaagaac agcagtgtgt tattatggct 180
gagaaccgta aatcctctat catcatccgt atgcgtgatg tggtactgtt tgagaaaaaa 240
gtctatctgt ctgagtgcaa aaccggtaac ggtaaaaact accgcggcac catgtccaaa 300
accaaaaacg gtattacttg ccagaaatgg tcctctactt ctccgcatcg tccacgtttc 360
tctccagcga ctcacccttc tgaaggtctg gaagagaact actgccgtaa cccggataac 420
gatccgcagg gtccgtggtg ttacactacc gacccggaaa aacgttacga ctactgcgat 480
atcctggaat gtgaagagga atgtatgcac tgctctggtg aaaactacga cggcaaaatc 540
agcaaaacca tgtctggcct ggagtgtcaa gcgtgggatt ctcagagccc gcatgcacac 600
ggttacatcc cgtccaaatt cccgaacaaa aacctgaaaa agaactattg ccgtaacccg 660
gatcgcgaac tgcgtccgtg gtgtttcacc accgacccga acaaacgttg ggaactgtgc 720
gatattccgc gttgtactac cccaccacct tcttctggcc caacttatca gtgtctgaaa 780
ggtaccggcg aaaactatcg cggcaacgtg gcagtgactg tatctggtca tacttgtcag 840
cactggagcg cacagacccc gcatacccat aaccgtaccc cggagaattt tccgtgtaaa 900
aacctggacg aaaactactg ccgtaaccct gacggtaaac gtgccccatg gtgtcacact 960
accaactccc aggtgcgttg ggaatactgc aaaatcccgt cctgtgatag ctctccggtt 1020
tctactgaac agctggcgcc tactgctcca ccggaactga ctccggtggt tcaggactgc 1080
taccacggtg atggccagtc ctaccgtggt acctcttcta ccaccactac cggtaaaaaa 1140
tgccagtcct ggagctctat gactccacac cgccaccaga aaacgccgga aaactaccca 1200
aatgccggcc tgaccatgaa ttactgtcgt aacccggatg cggacaaagg cccatggtgc 1260
ttcaccaccg acccaagcgt ccgttgggaa tactgtaacc tgaaaaagtg ctccggtacg 1320
gaagctagcg ttgttgcacc gccaccggtg gtactgctgc ctgatgtaga gaccccttct 1380
gaggaagatt gcatgttcgg taacggtaaa ggttaccgtg gcaaacgtgc taccaccgtt 1440
actggcactc cgtgtcagga ttgggcagct caggagccgc atcgtcactc tattttcacc 1500
ccagaaacga acccgcgcgc aggtctggaa aaaaactact gtcgtaaccc ggacggtgac 1560
gttggtggtc cgtggtgtta taccactaac ccgcgtaaac tgtatgatta ctgcgacgtt 1620
ccgcaatgtg ctgctccgtc ctttgactgc ggcaaaccgc aggtagaacc gaaaaaatgc 1680
ccgggtcgtg tagttggtgg ttgcgttgca cacccgcatt cctggccgtg gcaggtatct 1740
ctgcgtaccc gcttcggcat gcatttctgc ggtggcactc tgatctctcc ggaatgggtg 1800
ctgacggcag ctcactgtct ggaaaaatcc cctcgtccgt cctcctacaa agttattctg 1860
ggcgcgcatc aagaagtcaa tctggaaccg cacgtgcagg aaatcgaagt atctcgcctg 1920
tttctggaac cgactcgtaa agacatcgcg ctgctgaaac tgtctagccc ggcggtaatc 1980
accgataaag taatcccagc ttgcctgccg tctccgaact atgtagttgc cgaccgtacc 2040
gaatgcttca ttaccggctg gggtgaaacc cagggtactt tcggtgccgg tctgctgaaa 2100
gaagcgcaac tgccggtcat cgagaacaaa gtttgcaacc gttatgaatt tctgaacggc 2160
cgtgtccagt ctactgaact gtgcgctggt catctggcgg gtggtactga cagctgtcag 2220
ggtgactctg gtggtccact ggtttgtttt gaaaaagata aatacattct gcagggtgta 2280
acctcttggg gtctgggttg cgctcgtccg aacaaaccgg gtgtgtacgt gcgcgttagc 2340
cgtttcgtta cttggattga aggtgtgatg cgtaacaatg gcggcggtgg ttctggtggt 2400
ggtggttctg gtggcgaatt c 2421
<210> 11
<211> 1785
<212> DNA
<213> Artificial Sequence
<400> 11
ggatcctcct accaggtaat ctgccgcgac gaaaaaaccc agatgatcta ccagcagcat 60
cagtcctggc tgcgtccggt tctgcgctct aaccgtgttg aatattgctg gtgcaactcc 120
ggtcgtgcgc aatgccattc tgttccggtg aaatcttgct ctgagccacg ttgttttaac 180
ggtggtacct gccagcaggc actgtatttt tccgacttcg tatgtcagtg cccggaaggt 240
tttgcgggta aatgctgtga aatcgatacc cgtgctactt gctatgagga ccagggtatc 300
agctatcgtg gtacttggtc caccgctgaa agcggtgcgg aatgtactaa ctggaacagc 360
tccgctctgg cgcagaaacc gtactctggt cgtcgtcctg atgccatccg tctgggtctg 420
ggcaaccata actactgccg taacccggat cgtgatagca aaccgtggtg ttacgtattt 480
aaagctggta agtactcttc cgagttctgc agcacgccgg cgtgtagcga aggcaacagc 540
gattgctact tcggcaacgg ttccgcctac cgtggtaccc actccctgac cgaatctggc 600
gcaagctgtc tgccatggaa ctctatgatc ctgatcggta aggtctacac cgctcagaac 660
ccgtctgcgc aggctctggg tctgggtaag cacaactact gtcgtaaccc ggatggtgat 720
gctaaaccat ggtgccatgt tctgaaaaac cgtcgtctga cctgggaata ctgcgacgtg 780
ccgtcttgct ccacgtgcgg tctgcgtcag tactcccagc cgcagttccg cattaaaggt 840
ggtctgttcg ctgatattgc ctctcacccg tggcaggcag ctattttcgc caaacatcgt 900
cgttctccgg gtgaacgctt cctgtgcggt ggcatcctga tcagctcttg ctggattctg 960
tctgccgctc actgttttca ggaacgtttc ccaccacacc atctgaccgt tatcctgggc 1020
cgtacctacc gtgttgttcc gggtgaggag gaacagaaat tcgaggtgga gaaatacatt 1080
gtgcataaag agttcgacga tgatacctac gataacgaca tcgcactgct gcagctgaaa 1140
tccgactcta gccgctgtgc acaggagtcc agcgtggttc gtaccgtttg cctgcctccg 1200
gctgacctgc aactgcctga ctggaccgaa tgcgaactgt ctggttacgg caaacacgag 1260
gcactgagcc cgttctactc tgaacgtctg aaagaagcgc acgtgcgtct gtatccttct 1320
agccgctgta ccagccagca cctgctgaac cgtaccgtga ctgacaacat gctgtgcgca 1380
ggcgatactc gttctggcgg tccacaggct aacctgcacg atgcgtgtca gggtgattct 1440
ggcggtccgc tggtatgtct gaacgacggt cgtatgactc tggttggcat catctcctgg 1500
ggtctgggtt gcggtcagaa agacgtacca ggtgtttata ccaaagttac caactacctg 1560
gattggattc gtgataacat gcgtcctggt ggtggtggtt ctggtggtgg tggttctggc 1620
ggtgaattca gcaaaatgat cgaaggtgtt tttgcgaaag gtctgaaatt tgcatctcac 1680
ctgttcaaag gcatcggtgc aggtccgggt atcggtaaat tcctgcattc tgcgggcaaa 1740
tttggcaaag cgttcgtcgg cgaaatcatg aaatcttaaa agctt 1785
<210> 12
<211> 2577
<212> DNA
<213> Artificial Sequence
<400> 12
ggatccgaac cactggatga ctacgtaaat acgcagggtg cttccctgtt ctctgtaact 60
aaaaaacaac tgggcgctgg ttccattgaa gagtgcgcag ctaaatgtga agaggatgaa 120
gaatttacct gccgtgcttt ccagtaccac agcaaagaac agcagtgtgt tattatggct 180
gagaaccgta aatcctctat catcatccgt atgcgtgatg tggtactgtt tgagaaaaaa 240
gtctatctgt ctgagtgcaa aaccggtaac ggtaaaaact accgcggcac catgtccaaa 300
accaaaaacg gtattacttg ccagaaatgg tcctctactt ctccgcatcg tccacgtttc 360
tctccagcga ctcacccttc tgaaggtctg gaagagaact actgccgtaa cccggataac 420
gatccgcagg gtccgtggtg ttacactacc gacccggaaa aacgttacga ctactgcgat 480
atcctggaat gtgaagagga atgtatgcac tgctctggtg aaaactacga cggcaaaatc 540
agcaaaacca tgtctggcct ggagtgtcaa gcgtgggatt ctcagagccc gcatgcacac 600
ggttacatcc cgtccaaatt cccgaacaaa aacctgaaaa agaactattg ccgtaacccg 660
gatcgcgaac tgcgtccgtg gtgtttcacc accgacccga acaaacgttg ggaactgtgc 720
gatattccgc gttgtactac cccaccacct tcttctggcc caacttatca gtgtctgaaa 780
ggtaccggcg aaaactatcg cggcaacgtg gcagtgactg tatctggtca tacttgtcag 840
cactggagcg cacagacccc gcatacccat aaccgtaccc cggagaattt tccgtgtaaa 900
aacctggacg aaaactactg ccgtaaccct gacggtaaac gtgccccatg gtgtcacact 960
accaactccc aggtgcgttg ggaatactgc aaaatcccgt cctgtgatag ctctccggtt 1020
tctactgaac agctggcgcc tactgctcca ccggaactga ctccggtggt tcaggactgc 1080
taccacggtg atggccagtc ctaccgtggt acctcttcta ccaccactac cggtaaaaaa 1140
tgccagtcct ggagctctat gactccacac cgccaccaga aaacgccgga aaactaccca 1200
aatgccggcc tgaccatgaa ttactgtcgt aacccggatg cggacaaagg cccatggtgc 1260
ttcaccaccg acccaagcgt ccgttgggaa tactgtaacc tgaaaaagtg ctccggtacg 1320
gaagctagcg ttgttgcacc gccaccggtg gtactgctgc ctgatgtaga gaccccttct 1380
gaggaagatt gcatgttcgg taacggtaaa ggttaccgtg gcaaacgtgc taccaccgtt 1440
actggcactc cgtgtcagga ttgggcagct caggagccgc atcgtcactc tattttcacc 1500
ccagaaacga acccgcgcgc aggtctggaa aaaaactact gtcgtaaccc ggacggtgac 1560
gttggtggtc cgtggtgtta taccactaac ccgcgtaaac tgtatgatta ctgcgacgtt 1620
ccgcaatgtg ctgctccgtc ctttgactgc ggcaaaccgc aggtagaacc gaaaaaatgc 1680
ccgggtcgtg tagttggtgg ttgcgttgca cacccgcatt cctggccgtg gcaggtatct 1740
ctgcgtaccc gcttcggcat gcatttctgc ggtggcactc tgatctctcc ggaatgggtg 1800
ctgacggcag ctcactgtct ggaaaaatcc cctcgtccgt cctcctacaa agttattctg 1860
ggcgcgcatc aagaagtcaa tctggaaccg cacgtgcagg aaatcgaagt atctcgcctg 1920
tttctggaac cgactcgtaa agacatcgcg ctgctgaaac tgtctagccc ggcggtaatc 1980
accgataaag taatcccagc ttgcctgccg tctccgaact atgtagttgc cgaccgtacc 2040
gaatgcttca ttaccggctg gggtgaaacc cagggtactt tcggtgccgg tctgctgaaa 2100
gaagcgcaac tgccggtcat cgagaacaaa gtttgcaacc gttatgaatt tctgaacggc 2160
cgtgtccagt ctactgaact gtgcgctggt catctggcgg gtggtactga cagctgtcag 2220
ggtgactctg gtggtccact ggtttgtttt gaaaaagata aatacattct gcagggtgta 2280
acctcttggg gtctgggttg cgctcgtccg aacaaaccgg gtgtgtacgt gcgcgttagc 2340
cgtttcgtta cttggattga aggtgtgatg cgtaacaatg gcggcggtgg ttctggtggt 2400
ggtggttctg gtggcgaatt cagcaaaatg atcgaaggtg tttttgcgaa aggtctgaaa 2460
tttgcatctc acctgttcaa aggcatcggt gcaggtccgg gtatcggtaa attcctgcat 2520
tctgcgggca aatttggcaa agcgttcgtc ggcgaaatca tgaaatctta aaagctt 2577

Claims (10)

1.一种兼具防粘连功效的重组抗菌肽，其特征在于：所述重组抗菌肽由氨基酸序列如SEQ ID NO.1所示的抗菌肽与如下蛋白片段a或片段b连接而成；
片段a：氨基酸序列如SEQ ID NO.2所示；
片段b：氨基酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的兼具防粘连功效的重组抗菌肽，其特征在于：重组抗菌肽由抗菌肽与蛋白片段a或片段b通过连接肽连接而成，连接肽的氨基酸序列如SEQ ID NO.4所示，核苷酸序列如SEQ ID NO.5所示。
3.根据权利要求1所述的兼具防粘连功效的重组抗菌肽，其特征在于：所述重组抗菌肽的氨基酸序列如SEQ ID NO.6或SEQ ID NO.7所示。
4.一种编码权利要求1所述的兼具防粘连功效的重组抗菌肽的基因。
5.根据权利要求4所述的基因，其特征在于：所述基因包括核苷酸序列如SEQ ID NO.8所示的编码抗菌肽的基因和如下编码蛋白片段a的基因或编码蛋白片段b的基因连接而成：
编码蛋白片段a的基因的核苷酸序列如SEQ ID NO.9所示；
编码蛋白片段b的基因的核苷酸序列如SEQ ID NO.10所示。
6.根据权利要求4所述的基因，其特征在于：编码所述重组抗菌肽的核苷酸序列如SEQID NO.11或SEQ ID NO.12所示。
7.一种含有权利要求4-6任一所述的基因的表达载体。
8.根据权利要求7所述的表达载体，其特征在于，所述表达载体为Pet32A质粒。
9.一种含有权利要求4-6任一所述的基因的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌BL21。
10.一种权利要求1-3任一所述的兼具防粘连功效的重组抗菌肽在制备预防术后粘连及术后伤口被细菌感染药物中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482655A (zh) * 2009-06-26 2012-05-30 莱桑多公司 抗微生物剂
CN108840940A (zh) * 2018-06-29 2018-11-20 中国农业科学院饲料研究所 一种嵌合肽a6及其应用
CN113577014A (zh) * 2020-12-30 2021-11-02 广州图微科创生物科技有限公司 医疗器械、水凝胶及其制备方法与应用
CN113896768A (zh) * 2021-11-01 2022-01-07 深圳市维琪医药研发有限公司 抗菌肽以及它们的美容组合物或药用组合物和用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482655A (zh) * 2009-06-26 2012-05-30 莱桑多公司 抗微生物剂
CN108840940A (zh) * 2018-06-29 2018-11-20 中国农业科学院饲料研究所 一种嵌合肽a6及其应用
CN113577014A (zh) * 2020-12-30 2021-11-02 广州图微科创生物科技有限公司 医疗器械、水凝胶及其制备方法与应用
CN113896768A (zh) * 2021-11-01 2022-01-07 深圳市维琪医药研发有限公司 抗菌肽以及它们的美容组合物或药用组合物和用途

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Patentee after: Sensor Control Eagle Fly (Tianjin) Medical Technology Co.,Ltd.

Country or region after: China

Address before: 300450 Tianjin International Biomedical joint research laboratory building n1708-1, No. 220 Dongting Road, economic and Technological Development Zone, Binhai New Area, Tianjin

Patentee before: Tianjin Fuyuan Biomedical Technology Co.,Ltd.

Country or region before: China