CN110713531B - 蜱凋亡抑制蛋白iap及其基因和应用 - Google Patents

蜱凋亡抑制蛋白iap及其基因和应用 Download PDF

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CN110713531B
CN110713531B CN201910982130.2A CN201910982130A CN110713531B CN 110713531 B CN110713531 B CN 110713531B CN 201910982130 A CN201910982130 A CN 201910982130A CN 110713531 B CN110713531 B CN 110713531B
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周金林
玛依努尔·吐尔迪
张厚双
周勇志
曹杰
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Abstract

本发明公开了一种蜱凋亡抑制蛋白IAP，其具有SEQ ID NO.1所示的氨基酸序列。本发明还公开了蜱凋亡抑制蛋白IAP基因，其包含：编码SEQ ID NO.1所示氨基酸序列的核苷酸序列。本发明的蜱凋亡抑制蛋白IAP及其基因，适用于开发研制新型杀蜱生物制剂，具有广阔的应用前景。

Description

蜱凋亡抑制蛋白IAP及其基因和应用
技术领域
本发明涉及生物工程技术领域，尤其涉及一种蜱凋亡抑制蛋白IAP及其基因和应用。
背景技术
蜱是一类重要的吸血外寄生虫，绝大多数寄生在哺乳动物体表，少数寄生在鸟类、爬行类及两栖类。蜱不仅吸食大量血液，损伤宿主皮肤，而且是人和动物许多重要疾病的传播媒介。蜱的发育需要经过卵、幼蜱、若蜱及成蜱四个阶段。卵在适宜条件下孵出幼蜱，幼蜱吸血后蜕皮成为若蜱，若蜱吸血后蜕皮成为成蜱，雌性成蜱饱血后产卵。Rhipicephalushaemaphysaloides(镰形扇头蜱)是广泛分布于中国南部的蜱种之一，属于硬蜱家族，为三宿主蜱，生活史中的每个阶段都要更换宿主。成蜱吸血时间长3～10天，雄蜱可以重复间歇性的吸血，而雌蜱不可重复上体吸血。
蜱的唾液腺是蜱及蜱传病防治重要的靶器官，它参与抗凝血，免疫抑制及抗炎症作用来成功吸血和传播病原。雌蜱的唾液腺在蜱吸血末期和饱血后会发生快速的退化。蜱唾液腺退化机制的研究对探究雌蜱不能重复上体吸血的原因、蜱的生物防治，以及蜱传病原的传播机制具有重要意义。细胞凋亡被认为可能是蜱唾液腺退化的主要诱因，在哺乳动物和节肢动物昆虫中凋亡抑制蛋白IAPs被证实作用于细胞凋亡途径下游的caspases，是调控其是否能执行凋亡的分子开关，但在蜱中并没有该分子的相关报道，因此为了研究蜱唾液腺退化的机制，找到潜在的控制雌蜱唾液腺退化和吸血的分子靶标，选择对蜱凋亡抑制蛋白IAP的分子功能进行探究。
发明内容
本发明要解决的技术问题是提供一种蜱凋亡抑制蛋白IAP及其基因，该IAP基因可用于开发研制新型杀蜱生物制剂。
为了解决上述技术问题，本发明通过如下技术方案实现:
在本发明的一个方面，提供了一种蜱凋亡抑制蛋白IAP，其具有SEQ ID NO.1所示的氨基酸序列。
在本发明的另一方面，提供了一种蜱凋亡抑制蛋白IAP基因，其包含：编码SEQ IDNO.1所示氨基酸序列的核苷酸序列。
优选的，所述IAP基因的核苷酸序列如SEQ ID NO.2所示。
在本发明的另一方面，还提供了一种重组载体，包含上述蜱凋亡抑制蛋白IAP基因序列或其部分序列。
所述重组载体包括重组克隆载体或重组表达载体。
在本发明的另一方面，还提供了一种包含上述重组载体的宿主细胞。
在本发明的另一方面，还提供了一种双链RNA，所述双链RNA能够抑制上述蜱凋亡抑制蛋白IAP基因的表达。
优选的，所述双链RNA包含SEQ ID NO.15所示核苷酸序列或其互补序列。
在本发明的另一方面，还提供了一种包含上述双链RNA的杀蜱生物制剂。
在本发明的另一方面，还提供了一种上述蜱凋亡抑制蛋白IAP的应用，用于筛选防控蜱虫的生物制剂。
在本发明的另一方面，还提供了一种上述蜱凋亡抑制蛋白IAP基因在制备杀蜱生物制剂中的应用。
本发明蜱凋亡抑制蛋白IAP及其基因，经体内和体外RNA干扰实验表明，IAP干扰后抑制了蜱唾液腺在吸血前期的发育，造成其吸血障碍，适用于开发研制新型杀蜱生物制剂。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例2的重组蛋白Rh IAP的表达结果图；
图2是本发明实施例4的Rh IAP的体外RNA干扰实验结果图。
具体实施方式
下列实施例中，未注明具体条件的实验方法，通常按常规条件，如《分子克隆实验指南》(J.萨姆布鲁克，D.W.拉塞尔著，黄培堂，汪嘉玺，朱厚础，等译.第3版，北京：科学出版社，2002)中所述的方法进行。
本发明首次从镰形扇头蜱中获得一个新的凋亡抑制蛋白IAP基因，将该基因编码区克隆到表达载体，在大肠杆菌中进行重组表达，经体内和体外RNA干扰实验表明，IAP干扰后可以升高细胞凋亡的水平，加快蜱唾液腺的退化进程，进而造成蜱吸血障碍，非常适于开发研制新型杀蜱生物制剂。
实施例1基因Rh IAP的分子克隆和序列分析
1.材料与方法
1.1.蜱及实验动物
镰形扇头蜱采自湖北武昌，在本试验室经兔体人工饲养保存。6周龄雄性昆明系小白鼠购自上海杰思捷实验动物有限公司。新西兰大白兔购自松联实验动物饲养场。
1.2.细菌与质粒
质粒构建及蛋白表达用到的是大肠杆菌DH5α和BL21(DE3)感受态细胞(Transgen)。基因克隆载体为pMD-18T(Takara)，表达载体用的是pET30a vector(Novagen)，pCMV-HA(Takara)。
1.3.镰形扇头蜱IAP基因(Rh IAP)的分子克隆
TRIzol reagent(Invitrogen)法提取显微解剖后半饱血蜱的唾液腺RNA。通过去基因组反转录试剂盒从虫体的总RNA中获得去除基因组的cDNA，实验方法参照反转录试剂盒PrimeScriptTMRT reagent Kit with gDNA Eraser(TaKaRa,Dalian,China)说明书进行操作。根据转录组测序预测的IAP同源基因的序列设计R.hIAP的扩增引物是F:5’-TTGCCTGGAGCCTACGCAGTTGT-3’(SEQ ID NO.3)和R:5’-TACCCAAGGAGGGCAAA AGCCAACT-3’(SEQ ID NO.4)，纯化后的PCR扩增产物连接到克隆载体pMD-18T(Takara)中进行测序。
2.结果
R.hIAP基因全长2506bp(SEQ ID NO.2)，其ORF全长1887bp,编码628个氨基酸(SEQID NO.1)，预测的该蛋白的等电点为6.42，相对分子质量约为65.9kDa。氨基酸序列分析可知，该蛋白包含3个BIR结构域和一个在N末端的Ring指结构域，进化树分析可知，R.hIAP属于二型IAP2，相较于哺乳动物和肩突硬蜱，R.hIAP与节肢动物昆虫纲中蚁及蜂的亲源关系更近。
实施例2重组蛋白Rh IAP的表达与纯化
为达到较高的纯化水平，构建表达载体，使目的蛋白的N端具有His标签。将目的基因Rh IAP克隆到表达载体pET30a中，然后将载体转移到E.coli BL21。
根据pET30a vector载体序列及R.hIAP全长基因通过引物设计引入Bam HI和HindIII两个酶切位点以及部分载体序列的片段，方法参照In-Fusion HD Cloning Kits(Clontech,Takara Bio)试剂盒说明书，亚克隆到双酶切后的表达载体pET30a(Novagen)中，进行测序确保序列的准确性。将连接产物转化至感受态细胞E.coli BL21(DE3)中进行表达。表达菌经过1mM IPTG诱导，25℃孵育约16小时后收集菌体，保存在-80℃。重组蛋白通过Ni-NTA His·Bind Resin(Novagen)进行纯化，将收集的诱导后的菌体沉淀用bindingbuffer(NI-NTA Buffer Kit,Novagen)重悬，然后超声破碎，12,000×g，4℃离心10min，收集上清，纯化后进行SDS-PAGE凝胶电泳分析定量。
结果：重组蛋白成功地表达，分子量约为66kDa，与预测的大小一致(图1)。蛋白在上清表达，表达菌用1mM IPTG 25℃诱导16小时，然后进行纯化。
实施例3R.h IAP转录水平检测
根据经基因克隆及重测序的Rh IAP序列，利用Primer5.0软件设计荧光定量PCR特异性引物：F：5’-CTTCAGGCGTCCGAGGGAGAGC-3’(SEQ ID NO.5)和R：5’-ACCTTGTCTTGTTCGTGAATGTAGTAAAAGCCCG-3’(SEQ ID NO.6)，以延伸因子ELFIA为内参，每组检测重复三次，每次设三个平行重复，所得数据采用2ct方法分析基因转录水平。
结果：IAP在蜱的卵和幼虫阶段的转录水平高于若虫及成虫阶段，其中幼蜱的转录水平最高，而若蜱及成蜱无差别，不同性别也无差别；在不同器官中的转录水平从高到低依次是卵巢、血淋巴、中肠，唾液腺及脂肪体。在未吸血的唾液腺，以及吸血一、三、五，七天及饱血后一两天的唾液腺，除了在饱血当天转录水平有一个峰值，其他时间转录水平随着吸血天数逐渐降低。
实施例4R.h IAP的RNA干扰实验
1.R.h IAP的体内RNA干扰实验
根据Rh IAP的碱基序列，设计dsIAP(SEQ ID NO.15)、dsLuciferase合成的引物：
dsIAP-S1：5’-GGATCCTAATACGACTCACTATAGG CAGGCGTCCGAGGGAGAG-3’(SEQ IDNO.7)
dsIAP-A1:5’-AACGCTGTGGAGTCTGGTCGG-3’(SEQ ID NO.8)
dsIAP-S2:5’-CAGGCGTCCGAGGGAGAG-3’(SEQ ID NO.9)
dsIAP-A2:5’-GGATCCTAATACGACTCACTATAGGAACGCTGTGGAGTCTGGTCGG-3’(SEQ IDNO.10)
dsLuciferase S1:5’-GGATCCTAATACGACTCACTATAGGGCTTCCATCTTCCAGGGATAC-3’(SEQ ID NO.11)
dsLuciferase A1:5’-CGTCCACAAACACAACTCCTCC-3’(SEQ ID NO.12)
dsLuciferase S2:5’-GCTTCCATCTTCCAGGGATACG-3’(SEQ ID NO.13)
dsLuciferaseA2:5’-GGATCCTAATACGACTCACTATAGGCGTCCACAAACACAACTCCTC-3’(SEQ ID NO.14)
合成方法按照T7 RiboMAX Express RNAi System(promega)试剂盒说明书。选取120只未吸血的雌蜱，实验组显微注射dsIAP，每只1μg(60只)，对照组每只蜱注射等量的dsLuciferase(60只)，24h后观察其状态后，将蜱接种于新西兰大白兔上，观察蜱的上体及吸血情况。
结果：如下表1所示，同Luciferase对照组相比，注射等量的dsIAP的蜱不能正常吸血，出现吸血障碍最终死亡，但是对蜱的上体率并未产生显著影响,推测IAP干扰后抑制了蜱唾液腺在吸血前期的发育，造成其吸血障碍。
表1 R.h IAP的体内RNA干扰实验结果列表
Figure BDA0002235533990000051
2.Rh IAP的体外RNA干扰实验
收取吸血第5天的蜱20只，经显微解剖后收取其唾液腺，经L15培养基(1％penicillin)于24孔板中培养，每孔10只蜱的唾液腺，在实验组中加入dsIAP，使其终浓度为1μg/mL,对照组中加入等量的dsLuciferase。体外刺激24h后收集不同组的唾液腺样品进行western blot检测，qPCR检测。
结果：经R.hIAP的体外RNA干扰的唾液腺组织，制作切片后Tunel染色检测可知，dsIAP组的凋亡程度显著高于对照组(图2)。IAP干扰后可以升高细胞凋亡的水平，一定程度上加快蜱唾液腺的退化进程。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 中国农业科学院上海兽医研究所（中国动物卫生与流行病学中心上海分中心 ）
<120> 蜱凋亡抑制蛋白IAP及其基因和应用
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<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 镰形扇头蜱(Rhipicephalus haemaphysaloide)
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370 375 380
Thr Lys Cys Phe His Cys Asp Gly Gly Leu Cys Asn Trp Asp Ser Asn
385 390 395 400
Asp Asp Pro Trp Glu Glu His Ala Arg Trp Phe Pro Arg Cys Gln Phe
405 410 415
Val Leu Leu Ser Lys Gly Glu Thr Tyr Val Gln Asp Cys Leu Arg Arg
420 425 430
His Gln Ser His Leu Ser Ala Val Ala Ala Ser Ala Ser Thr Ser Ser
435 440 445
Gln Gly Gln Ser Gly Gly Ala Asp Glu Gly Met Ala Thr Glu Leu Ala
450 455 460
Ala Leu Met Arg Ser Glu Asp Val Gln Phe Tyr Leu Ser Gln Gly Val
465 470 475 480
Pro Ala Glu Thr Leu Arg Ala Ala Leu Leu Lys His Met Arg Ser Gln
485 490 495
Gly Arg Gly Phe Ala Ser Arg Asp Glu Leu Leu Gln Val Leu Gly Glu
500 505 510
Leu Leu Thr Leu Pro Arg Ala Ser Ala Asp Gln Thr Pro Gln Arg Ser
515 520 525
Ser Thr Asn Ser Val Thr Ile Thr Lys Val Ala Thr Asn Pro Gln Ala
530 535 540
Asp Ser Val Val Ser Arg Ala Lys Asn Ala Ser Ala Glu Ser Leu Thr
545 550 555 560
Glu Gly Ser Glu Ser Ser Asp Leu Ala Leu Glu Asn Leu Arg Leu Lys
565 570 575
Asp Gln Arg Leu Cys Lys Val Cys Leu Asp Ala Glu Val Gly Val Val
580 585 590
Phe Leu Pro Cys Gly His Leu Val Ala Cys Pro Ala Cys Ala Ser Ala
595 600 605
Leu Ser Asp Cys Pro Val Cys Arg Ala Ser Ile Arg Gly Thr Val Arg
610 615 620
Thr Phe Phe Ser
625
<210> 2
<211> 2506
<212> DNA
<213> 镰形扇头蜱(Rhipicephalus haemaphysaloide)
<220>
<221> CDS
<222> (276)..(2162)
<400> 2
tacgccaagc ttgcatgcct gcaggtcgac gattttgcct ggagcctacg cagttgtgaa 60
acaggaggaa taccaggagg tgcgccttgg ttgcgttagg tttcccaagt tgaatttcgt 120
tgctgttcac tcggctccgc tgtttcgtct ggacaggatc ggcctgtgtg gggccagtca 180
tcacgcagag aggcgtgtac gatggataag cctagagcca atgcgagtgg tacaggaaat 240
cagcgtgctt tatcaatgac gcggcggtaa aatga aat ttc ctg ata acc att cac 296
Asn Phe Leu Ile Thr Ile His
1 5
tgc tca gtt cgg cgt ccc gac tgc cgc aca tcg agc ctg ttg atg tgt 344
Cys Ser Val Arg Arg Pro Asp Cys Arg Thr Ser Ser Leu Leu Met Cys
10 15 20
tca atg gcg tct atg atg gtg ggc acg gca gtg acg caa cgg ggg ccg 392
Ser Met Ala Ser Met Met Val Gly Thr Ala Val Thr Gln Arg Gly Pro
25 30 35
ctc gcc ttc ccg gca tct gag cag acg gtt ctg gat ggg tcc atg ggg 440
Leu Ala Phe Pro Ala Ser Glu Gln Thr Val Leu Asp Gly Ser Met Gly
40 45 50 55
gca gtg gtg agc aag ttc acc aag atg gcc ggt act gct gac tat agc 488
Ala Val Val Ser Lys Phe Thr Lys Met Ala Gly Thr Ala Asp Tyr Ser
60 65 70
cga gag gaa cac agg ctt ccc act ttt agt gag tgg ccc cca aac gcc 536
Arg Glu Glu His Arg Leu Pro Thr Phe Ser Glu Trp Pro Pro Asn Ala
75 80 85
cca gta tct gcg aaa aag ctg gcc cag ggt ggc ttt gtg tgt att aac 584
Pro Val Ser Ala Lys Lys Leu Ala Gln Gly Gly Phe Val Cys Ile Asn
90 95 100
gct tcc aga ctt ctg aca cgg tgt tac agt tgt ggg ctt gag gtt tca 632
Ala Ser Arg Leu Leu Thr Arg Cys Tyr Ser Cys Gly Leu Glu Val Ser
105 110 115
gac tgg cgt atc gca gat gtt gtg gta gac aag cac cgg acg atg cgg 680
Asp Trp Arg Ile Ala Asp Val Val Val Asp Lys His Arg Thr Met Arg
120 125 130 135
cca gac tgt gcg ttt gtg cgc tcc ctt cca agt ccg cat gtt tcg ggg 728
Pro Asp Cys Ala Phe Val Arg Ser Leu Pro Ser Pro His Val Ser Gly
140 145 150
gca cca agc cct cag tcg ccc acg acc atc cct cca tct gcg ggc gca 776
Ala Pro Ser Pro Gln Ser Pro Thr Thr Ile Pro Pro Ser Ala Gly Ala
155 160 165
agg cat gtg caa gcg atg cac gtt gac agt gtg gct cct cca tta ccc 824
Arg His Val Gln Ala Met His Val Asp Ser Val Ala Pro Pro Leu Pro
170 175 180
tgc atc aac agt acg cct tca gtg caa agt cgg tct tgt gag act gtg 872
Cys Ile Asn Ser Thr Pro Ser Val Gln Ser Arg Ser Cys Glu Thr Val
185 190 195
ccg cag agt ggt gca cga aat gac atc ccg ctt cat cgg ctt cag gcg 920
Pro Gln Ser Gly Ala Arg Asn Asp Ile Pro Leu His Arg Leu Gln Ala
200 205 210 215
tcc gag gga gag cgg ttc aac acg ttt tat gac tgg ccg ctg gtc tct 968
Ser Glu Gly Glu Arg Phe Asn Thr Phe Tyr Asp Trp Pro Leu Val Ser
220 225 230
ccg tca cca cgg gct ctc gcg cag gcg ggc ttt tac tac att cac gaa 1016
Pro Ser Pro Arg Ala Leu Ala Gln Ala Gly Phe Tyr Tyr Ile His Glu
235 240 245
caa gac aag gtg cag tgt gcc ttc tgc aag ggc ata gtg cac aac tgg 1064
Gln Asp Lys Val Gln Cys Ala Phe Cys Lys Gly Ile Val His Asn Trp
250 255 260
gag cca ggg gac gac ccc ctg cgg gag cat gcc cac cac tac ccg tgt 1112
Glu Pro Gly Asp Asp Pro Leu Arg Glu His Ala His His Tyr Pro Cys
265 270 275
tgc cgg tat ctt ctg gac cct gac cta gct ggc cat gac gag tgc ggt 1160
Cys Arg Tyr Leu Leu Asp Pro Asp Leu Ala Gly His Asp Glu Cys Gly
280 285 290 295
cac atg tcg tgg cag cag gcg cgc tcc gca ccc gag tgt gcg cag ctg 1208
His Met Ser Trp Gln Gln Ala Arg Ser Ala Pro Glu Cys Ala Gln Leu
300 305 310
ctc atc agc gaa ggc ccc cag gga gtg cac ttg aaa gga aac gct cct 1256
Leu Ile Ser Glu Gly Pro Gln Gly Val His Leu Lys Gly Asn Ala Pro
315 320 325
cca tca gag ctg tct ggc ctc ggc gtg tcc gtg cac gtc gga cca aag 1304
Pro Ser Glu Leu Ser Gly Leu Gly Val Ser Val His Val Gly Pro Lys
330 335 340
cat ccc tcg caa gca tcg ccc gac gcc cga ctg cgg act tac gac aag 1352
His Pro Ser Gln Ala Ser Pro Asp Ala Arg Leu Arg Thr Tyr Asp Lys
345 350 355
tgg ccc acg acg tgc ccc aag cgg ccc cag gaa tta gta gtg gct gga 1400
Trp Pro Thr Thr Cys Pro Lys Arg Pro Gln Glu Leu Val Val Ala Gly
360 365 370 375
ttt ttt tac att ggc gtt aat gat tat acc aag tgc ttc cac tgt gat 1448
Phe Phe Tyr Ile Gly Val Asn Asp Tyr Thr Lys Cys Phe His Cys Asp
380 385 390
ggt ggc ctc tgc aat tgg gac tcg aat gac gac ccc tgg gag gaa cac 1496
Gly Gly Leu Cys Asn Trp Asp Ser Asn Asp Asp Pro Trp Glu Glu His
395 400 405
gcc cgt tgg ttt cct cgt tgc cag ttt gtg ctg ctg tcc aag ggc gag 1544
Ala Arg Trp Phe Pro Arg Cys Gln Phe Val Leu Leu Ser Lys Gly Glu
410 415 420
aca tac gtg cag gac tgc ctc agg cga cac cag tcg cac ctc agc gcg 1592
Thr Tyr Val Gln Asp Cys Leu Arg Arg His Gln Ser His Leu Ser Ala
425 430 435
gtg gcg gcc agt gcg tca aca agt tcc cag gga cag tcc gga ggt gca 1640
Val Ala Ala Ser Ala Ser Thr Ser Ser Gln Gly Gln Ser Gly Gly Ala
440 445 450 455
gac gag gga atg gcg acc gaa ctg gca gcg ttg atg cga tcc gaa gat 1688
Asp Glu Gly Met Ala Thr Glu Leu Ala Ala Leu Met Arg Ser Glu Asp
460 465 470
gtg cag ttc tac ttg tca caa ggg gtc cct gca gag aca ctg cgc gca 1736
Val Gln Phe Tyr Leu Ser Gln Gly Val Pro Ala Glu Thr Leu Arg Ala
475 480 485
gcg ttg ctt aag cac atg cgc agt cag gga cgt ggt ttt gcg agc cgg 1784
Ala Leu Leu Lys His Met Arg Ser Gln Gly Arg Gly Phe Ala Ser Arg
490 495 500
gat gag ctt ctt cag gtt ctt ggc gag ctg ctt acc cta cca agg gct 1832
Asp Glu Leu Leu Gln Val Leu Gly Glu Leu Leu Thr Leu Pro Arg Ala
505 510 515
tca gcc gac cag act cca cag cgt tcg tct aca aac agt gtc acg atc 1880
Ser Ala Asp Gln Thr Pro Gln Arg Ser Ser Thr Asn Ser Val Thr Ile
520 525 530 535
acc aaa gtg gca aca aat cct cag gcg gat agc gta gtt tct aga gca 1928
Thr Lys Val Ala Thr Asn Pro Gln Ala Asp Ser Val Val Ser Arg Ala
540 545 550
aag aat gct tca gcg gag agc ctt aca gaa ggg tct gag tct tct gat 1976
Lys Asn Ala Ser Ala Glu Ser Leu Thr Glu Gly Ser Glu Ser Ser Asp
555 560 565
ttg gct ctg gag aac ctg cga ctc aag gac cag cgc ctc tgc aag gtg 2024
Leu Ala Leu Glu Asn Leu Arg Leu Lys Asp Gln Arg Leu Cys Lys Val
570 575 580
tgt ctc gat gcc gag gta ggc gtc gtg ttc ctg ccg tgc ggc cac ctg 2072
Cys Leu Asp Ala Glu Val Gly Val Val Phe Leu Pro Cys Gly His Leu
585 590 595
gtg gcg tgc ccc gct tgc gcc tcc gcc ctc tcc gac tgc cca gtg tgc 2120
Val Ala Cys Pro Ala Cys Ala Ser Ala Leu Ser Asp Cys Pro Val Cys
600 605 610 615
cgc gcg tcc atc cgc ggc acc gtg cgg acg ttc ttc tcg tga aatcgatggc 2172
Arg Ala Ser Ile Arg Gly Thr Val Arg Thr Phe Phe Ser
620 625
agagagagaa ggattgtgca tgagatacgc cgctgcaaca gtgtcttcag caacgttgta 2232
gtttaggaga cgcaacagcc cttgatacac aattaggaaa tgcttcaaaa aataatttcc 2292
cgccttttta tctgtctagt aacttgtttt aatttgcaac gctcgattcg gtaagacccc 2352
atgctaaggc atacctacgc tggtaaactg tacggagagc ttgaagtgcg tgcctgctga 2412
ttttaatgtg gctgcagtag aatacaagtt ggcttttgcc ctccttgggt aaatctctag 2472
aggatccccg ggtaccgagc tcgaattcac tggc 2506
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial)
<400> 3
ttgcctggag cctacgcagt tgt 23
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 4
tacccaagga gggcaaaagc caact 25
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 5
cttcaggcgt ccgagggaga gc 22
<210> 6
<211> 34
<212> DNA
<213> 人工序列(Artificial)
<400> 6
accttgtctt gttcgtgaat gtagtaaaag cccg 34
<210> 7
<211> 43
<212> DNA
<213> 人工序列(Artificial)
<400> 7
ggatcctaat acgactcact ataggcaggc gtccgaggga gag 43
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 8
aacgctgtgg agtctggtcg g 21
<210> 9
<211> 18
<212> DNA
<213> 人工序列(Artificial)
<400> 9
caggcgtccg agggagag 18
<210> 10
<211> 46
<212> DNA
<213> 人工序列(Artificial)
<400> 10
ggatcctaat acgactcact ataggaacgc tgtggagtct ggtcgg 46
<210> 11
<211> 46
<212> DNA
<213> 人工序列(Artificial)
<400> 11
ggatcctaat acgactcact atagggcttc catcttccag ggatac 46
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 12
cgtccacaaa cacaactcct cc 22
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 13
gcttccatct tccagggata cg 22
<210> 14
<211> 46
<212> DNA
<213> 人工序列(Artificial)
<400> 14
ggatcctaat acgactcact ataggcgtcc acaaacacaa ctcctc 46
<210> 15
<211> 943
<212> RNA
<213> 人工序列(Artificial)
<400> 15
caggcguccg agggagagcg guucaacacg uuuuaugacu ggccgcuggu cucuccguca 60
ccacgggcuc ucgcgcaggc gggcuuuuac uacauucacg aacaagacaa ggugcagugu 120
gccuucugca agggcauagu gcacaacugg gagccagggg acgacccccu gcgggagcau 180
gcccaccacu acccguguug ccgguaucuu cuggacccug accuagcugg ccaugacgag 240
ugcggucaca ugucguggca gcaggcgcgc uccgcacccg agugugcgca gcugcucauc 300
agcgaaggcc cccagggagu gcacuugaaa ggaaacgcuc cuccaucaga gcugucuggc 360
cucggcgugu ccgugcacgu cggaccaaag caucccucgc aagcaucgcc cgacgcccga 420
cugcggacuu acgacaagug gcccacgacg ugccccaagc ggccccagga auuaguagug 480
gcuggauuuu uuuacauugg cguuaaugau uauaccaagu gcuuccacug ugaugguggc 540
cucugcaauu gggacucgaa ugacgacccc ugggaggaac acgcccguug guuuccucgu 600
ugccaguuug ugcugcuguc caagggcgag acauacgugc aggacugccu caggcgacac 660
cagucgcacc ucagcgcggu ggcggccagu gcgucaacaa guucccaggg acaguccgga 720
ggugcagacg agggaauggc gaccgaacug gcagcguuga ugcgauccga agaugugcag 780
uucuacuugu cacaaggggu cccugcagag acacugcgcg cagcguugcu uaagcacaug 840
cgcagucagg gacgugguuu ugcgagccgg gaugagcuuc uucagguucu uggcgagcug 900
cuuacccuac caagggcuuc agccgaccag acuccacagc guu 943

Claims (10)

1.一种蜱凋亡抑制蛋白IAP，其氨基酸序列如SEQ ID NO.1所示。
2.一种蜱凋亡抑制蛋白IAP基因，其基因序列为编码SEQ ID NO.1所示氨基酸序列的核苷酸序列。
3.根据权利要求2所述的蜱凋亡抑制蛋白IAP基因，其特征在于，所述IAP基因的核苷酸序列如SEQ ID NO.2所示。
4.一种重组载体，其特征在于，包含：权利要求2所述的蜱凋亡抑制蛋白IAP基因序列。
5.一种宿主细胞，其特征在于，包含权利要求4所述的重组载体。
6.一种双链RNA，其特征在于，所述双链RNA能够抑制权利要求2或3所述蜱凋亡抑制蛋白IAP基因的表达。
7.根据权利要求6所述的双链RNA，其特征在于，所述双链RNA包含SEQ ID NO.15所示核苷酸序列或其互补序列。
8.权利要求1所述蜱凋亡抑制蛋白IAP的应用，用于筛选防控蜱虫的生物制剂。
9.权利要求2所述蜱凋亡抑制蛋白IAP基因在制备杀蜱生物制剂中的应用。
10.一种杀蜱生物制剂，其特征在于，包含权利要求6或7所述的双链RNA。
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