CN106518995B - 印鼠客蚤多肽及其基因和应用 - Google Patents
印鼠客蚤多肽及其基因和应用 Download PDFInfo
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Abstract
本发明涉及一种印鼠客蚤多肽及其基因和应用,具体涉及印鼠客蚤多肽FS48在制备治疗Kv1.1相关的疼痛、癌症、炎性疾病、神经退行性疾病、心血管疾病、自免疫疾病和慢性感染性疾病药物中应用,其中所述的印鼠客蚤多肽FS48的氨基酸序列如SEQ ID NO:1所示。本发明所多肽由以下方法制备得:原核表达印鼠客蚤多肽FS48,然后采用三步柱层析分离纯化后得到。本发明所述的印鼠客蚤多肽FS48能通过抑制Kv1.1和Kv4.1而具有镇痛、抗炎、免疫调节和抗肿瘤作用,且无细胞毒性。
Description
技术领域:
本发明涉及生物医学领域,具体涉及一种从动物组织得到的蛋白及其用途。
背景技术:
钾通道是广泛分布于骨骼肌、神经、心脏、血管、腺体等细胞和组织上的一类跨膜蛋白,其在许多细胞兴奋性和非兴奋性细胞的信号传递过程中有重要作用,如,动作电位调节、心脏起搏、信号整合、激素释放、细胞容积调节和细胞增殖等方面(Curr Opin SupportPalliat Care,2015Jun;9(2):147-54)。此外,钾通道在前列腺癌、结肠癌、肺癌和乳腺癌的细胞增殖和生长中的作用也被发现(Annu Rev Cell Dev Biol,2015;31:231-47)。目前已经有大约80种不同的钾通道亚型被报到,其中Kv1.1在CD4+辅助性T细胞、血管平滑肌细胞、中枢和外周神经元细胞以及肺癌细胞和乳腺癌细胞上表达,其与细胞炎性因子分泌、疼痛信号传导、血管重塑等过程密切相关。因此,Kv1.1是治疗疼痛、癌症、炎性疾病、神经退行性疾病、心血管疾病、自免疫疾病和慢性感染性疾病的靶点(Pharmacol Ther,2016,159:93-101;Trends Neurosci,2014,37(3):146-58)。例如,抑制Kv1.1能降低A549细胞的增殖使细胞处在细胞周期的静息期;麻醉剂氨基苯甲酸丁酯、布比卡因、丁哌卡因、罗哌卡因和马比佛卡因能抑制Kv1.1具有镇痛作用(Anesthesiology,2008,109(5):895-904)。
有毒动物,如蝎、蛇、蜘蛛、蟾蜍、蜈蚣等的毒素是我国传统医药中治疗疑难杂症重要源泉。动物多肽毒素已成为全球研发相关疾病药物的重要资源。例如,中国科学院、军事医学科学院、湖南师范大学等单位的研究人员分别从眼镜蛇、芋螺、蜘蛛的毒素中发现的cobra toxin、SO-3和HWAP-I作为镇痛药物被广泛应用于临床(Expert Opin Biol Ther,2011,11(11):1469-84);而美国FDA已批准来源于蜥蜴和芋螺毒素的Exenatide和Ziconotide分别用于临床治疗糖尿病和疼痛(Methods Find Exp Clin Pharmacol,2009,31(7):463-93)。另外,上海大学相关实验室针对东亚钳蝎多肽毒素的系统动物疼痛或镇痛行为模型研究表明我国蝎毒中含有多种致痛/镇痛或癫痫/抗癫痫的膜钠或钾通道活性成份(J Neurooncol,2005,73(1):53-56;J Clin Oncol,2006,24(22):3644-3650)。因此,动物毒素将是研发新药的有效途径。
我国对蝎、蛇、蜘蛛、蟾蜍、蜈蚣等的毒素的药用研究有悠久的历史,但对跳蚤等吸血节肢动物体内药理活性组分研究不多。吸血节肢动物通常需要在生命周期内的一定时期吸血以完成生命循环。在长期的吸血进化过程中,吸血节肢动物在其唾液内形成了众多的药理活性物质以克服宿主的防御。如,牛虻的唾液腺中含有许多血小板聚集抑制因子、抗血浆凝集因子、血管舒张剂和免疫调节肽等。因此牛虻成为活血化瘀中药材(Mol CellProteomics,2008,7(3):582-90)。印鼠客蚤(Xenopsylla cheopis)是我国常见的吸血昆虫,分布于除新疆、西藏和宁夏外的广大地区。它们只有从小家鼠、褐家鼠、黄胸鼠和人身上吸血后才能产卵完成生命周期。
发明人将本发明的印鼠客蚤多肽FS48在pubmed数据库进行搜寻比较,未发现有任何关于该蛋白功能的报道。
发明内容:
为了解决上述问题,本发明一个方面提供了一种印鼠客蚤多肽FS48,其序列如SEQID No.1所示。
本发明另一个方面提供了编码如前1所述的多肽的核苷酸。
本发明再一个方面提供了一种药物组合物,其包含权利要求1所述的多肽和/或权利要求2所述的核苷酸,以及可药用的辅料。
其中,所述药物组合物其制剂为注射剂。
本发明再一个方面提供了一种前述的印鼠客蚤多肽FS48的制备方法,该方法包括以下步骤:
(1)提取印鼠客蚤唾液腺总mRNA,并采用SEQ ID NO:2所示的上游引物和SEQ IDNO:3所示的下游引物扩增编码SEQ ID NO:4所示的印鼠客蚤抗心律失常的基因,然后先将该基因克隆到pET-17b表达载体中,再转化到BL21(DE3)pLysS大肠杆菌中重组表达,并收集目的细胞超声上清;
(2)将所收集的上清透析后依次上Hiprep SP阳离子交换色谱柱、Sephadex SR-100凝胶过滤柱、Hiprep SP阳离子交换色谱柱和SephadexTM G75 10/300GL凝胶过滤柱层析,得到如SEQ ID NO:1所示的印鼠客蚤唾液腺基因重组表达蛋白,即印鼠客蚤多肽FS48。
本发明一个方面提供了前述的多肽或者前述的药物组合物在制备抗肿瘤的药物中的用途。
本发明一个方面提供了前述的多肽或者前述的药物组合物在制备镇痛和/或抗炎的药物中的用途。
本发明一个方面提供了前述的多肽或者前述的药物组合物在制备治疗神经退行性疾病或心脑血管疾病或自免疫疾病或慢性感染疾病的药物中的用途。
本发明一个方面提供了前述的多肽或者前述的药物组合物在制备靶向Kv1.1的药物中的用途。
多肽多肽多肽多肽本发明的印鼠客蚤唾液腺多肽FS48,对大鼠背根神经节上的钾离子通道有明显的抑制作用,并且该毒素能够选择性的作用钾离子通道亚型Kv1.1、Kv1.2和Kv4.1。印鼠客蚤唾液腺多肽FS48在小鼠的疼痛模型及炎症模型上表明出很好的治疗效果,能显著影响小鼠脾细胞增殖和细胞因子分泌,且能抑制非小细胞肺癌细胞NCI-H460的迁移。因此印鼠客蚤唾液腺多肽FS48能够在治疗K1.1相关的疼痛、癌症、炎性疾病、神经退行性疾病、心血管疾病、自免疫疾病和慢性感染性疾病的药物中得到应用。
附图说明:
图1为本发明印鼠客蚤多肽FS48大肠杆菌表达结果电泳图。从左到有泳道分别是蛋白质标准,37℃1.0mM IPTG诱导样品,37℃0.4mM IPTG诱导样品,30℃0.4mM IPTG诱导样品,箭头所示为目的带。
图2为本发明印鼠客蚤多肽FS48的分离纯化图,图中,箭头所示为目的峰,A、B、C和D分别是利用Hiprep SP阳离子交换、Sephadex SR-100凝胶过滤、Hiprep SP阳离子交换和SephadexTM G75 10/300GL凝胶过滤色谱柱层析后得到的图,其中图2D中电泳图是SephadexTM G75 10/300GL凝胶过滤层析后得到的纯蛋白电泳结果,图中条带为目的蛋白,其被切胶用于N末端测序。
图3为本发明印鼠客蚤多肽FS48膜片钳分析结果图。A,样品对DRG上钾电流的影响;B,为样品对表达Kv4.1哺乳动物细胞钾电流的影响;C,为样品对表达Kv2.1哺乳动物细胞钾电流的影响;D,为样品对表达Kv1.1哺乳动物细胞钾电流的影响;E,为样品对Kv1.1通道抑制的浓度依赖曲线;F,为样品对Kv1.1通道稳定活化的影响。
图4为本发明印鼠客蚤多肽FS48的镇痛效果。A为鼠尾热痛实验结果;B为福尔马林致痛实验结果;C为醋酸扭体实验结果。
图5为本发明印鼠客蚤多肽FS48的抗炎效果。A为FS48对小鼠脚爪肿胀体积的影响,B为FS48对小鼠脚爪髓过氧化物酶活性的影响。
图6为本发明印鼠客蚤多肽FS48抗肿瘤划痕实验结果。A为FS48对肿瘤细胞迁移的影响,B为统计学分析。
图7为本发明印鼠客蚤多肽FS48抗肿瘤侵袭实验结果,A为FS48对肿瘤细胞侵袭的影响,B为统计学分析。
图8为本发明印鼠客蚤多肽FS48对小鼠脾细胞增殖和细胞因子分泌的影响。A为对小鼠脾细胞增殖的影响,B为对小鼠脾细胞分泌IL-2的影响,C为对小鼠脾细胞分泌INF-γ的影响,D为对小鼠脾细胞分泌TNF-α的影响。
具体实施方式:
印鼠客蚤多肽FS48注射液的制备:
实施例1印鼠客蚤多肽FS48基因克隆
I、印鼠客蚤唾液腺总RNA提取:活体印鼠客蚤放在冰上30-60min冰冻麻醉,在显微镜下解剖50只印鼠客蚤得到唾液腺,加入1mL总RNA提取缓冲液(Trizol溶液,美国GIBCOBRL公司产品),于5m1玻璃匀浆器中匀浆1分钟。加入等体积酚/氯仿溶液,剧烈混匀,室温放置1分钟,4℃,12000rpm离心10分钟,弃除沉淀。上清加入等体积的异丙醇,室温放置4分钟,4℃,12000rpm离心10分钟,沉淀用75%乙醇洗一次,晾干,管底沉淀物即为印鼠客蚤唾液腺总RNA。
II、印鼠客蚤唾液腺mRNA的纯化:印鼠客蚤唾液腺mRNA分离纯化采用美国PROMEGA公司的mRNA Isolation Systems试剂盒。取印鼠客蚤唾液腺总RNA 500μg溶于500μl DEPC水中,放入65℃水浴10分钟,加入3μl的Oligo(dT)探针和13μl 20×SSC溶液,混匀,放置室温冷却,称为A液。磁珠(SA-PMP)的洗涤:将磁珠轻弹混匀,至磁力架吸附30秒,弃上清,加0.5×SSC 0.3m1,至磁力架吸附30秒,最后加0.1ml 0.5×SSC悬浮,称之为B液。将A液加入B液中,室温放置10分钟,至磁力架吸附30秒,弃上清,用0.1×SSC洗涤4次,最后弃上清,加0.l ml DEPC水悬浮,至磁力架上吸附30秒,将上清移至新的试管,再加入0.15m1DEPC水重新悬浮,至磁力架吸附30秒,移上清至上述试管,则上清中为纯化的印鼠客蚤唾液腺mRNA。加入1/10体积3M乙酸钠,pH 5.2,等体积异丙醇,于-70℃放置30分钟,4℃,12000rpm离心10分钟,弃上清,沉淀溶解于10μl DEPC水中。
III、印鼠客蚤唾液腺总cDNA扩增:采用CLONTECH公司CreatorTM SMART TM cDNALibrary Construction Kit质粒cDNA文库构建试剂盒扩增。A.cDNA第一链合成(mRNA反转录):在0.5ml无菌的离心管加入1μl印鼠客蚤唾液腺mRNA、1μl SMART IV寡聚核苷酸、1μlCDS III/3’PCR引物,加2μl去离子水使总体积达到5μl。混匀离心管中的试剂并以12000rpm离心15秒,72℃保温2分钟。将离心管在冰上孵育2分钟。在离心管中加入2.0μl 5×第一链缓冲、1.0μl 20mM二硫苏糖醇、1.0μl 10mM dNTP混合物、1.0μl PowerScript反转录酶。混合离心管中试剂并以12000rpm离心15秒,在42℃保温1小时。将离心管置于冰上中止第一链的合成。从离心管取2μl所合成的cDNA第一链备用。B.采用长末端聚合酶链式反应(LD-PCR)方法扩增第二链:95℃预热PCR仪。将2μl cDNA第一链(mRNA反转录)、80μl去离子水、10μl10×Advantage 2PCR缓冲、2μl 50×dNTP混合物、2μl 5’PCR引物、2μl CDS III/3’PCR引物以及2μl大肠杆菌聚合酶离心管进行反应。在PCR仪中按以下程序扩增:①95℃,20秒钟;②22个循环:95℃,5秒钟;68℃,6分钟。循环结束后,将离心管中合成的cDNA双链进行抽提。C.PCR产物用PROMEGA公司的SV Gel and PCR Clean-Up System试剂盒进行抽提回收,步骤如下:将通过PCR得到的cDNA双链加入等体积的膜结合缓冲颠倒混匀,然后将混和液转入离心纯化柱,室温静置5分钟,使DNA充分与硅胶膜结合。以12000rpm离心30秒,倒掉收集管中的废液。加入700μl的洗脱液(含乙醇)于离心纯化柱中,以12000rpm离心30秒,倒掉收集管中的废液。重复步骤2,12000rpm离心5分钟。将离心纯化柱置于新的离心管中,加入30μl超纯水,在室温下静置5分钟,以12000rpm离心30秒,管底溶液即为所纯化过的印鼠客蚤唾液腺总cDNA。
Ⅳ、印鼠客蚤唾液腺多肽FS48基因的克隆:依据报道的印鼠客蚤唾液腺核苷酸序列(GenBank:EF179444.1)为参考设计引物,正向引物序列为5’GGCATATGATAAGTGCAACTGAGTATAAATGC3’(SEQ ID NO:2),反向引物序列为5’CTCGAGACAATAGCATTTCTTTCC 3’(SEQ IDNO:3)。以100倍稀释纯化过的印鼠客蚤唾液腺总cDNA为模板扩增印鼠客蚤唾液腺多肽FS48的基因。PCR反应程序为:①95℃,4分钟;②35个循环:94℃,30秒钟;56℃,30秒钟;72℃,45秒钟;③72℃,10分钟。将扩增产物用1%琼脂糖凝胶电泳,切取目的条带,用DNA纯化试剂盒进行纯化。将纯化的目的片段连接到pMD19-T载体中,即得连接产物。将连接产物转化进Takara公司的大肠杆菌DH5α感受态细胞。取适量转化产物涂布至含Amp的LB平板上,经37℃培养16小时形成的单菌落即为含目的基因的阳性克隆,提取质粒酶切鉴定并送到英伟创津公司测序。测序结果显示基因自5’端至3’端序列为(SEQ ID NO:4):
CATATGATAAGTGCAACTGAGTATAAATGCAAAGTAACAGGAACAGGTGATATGACAATACCATTTGGATGTAAAGATGGCAATGATGTTCAAGGATGTAAAAAACTTTGTCAGGAGAAATGTAAATACACGACAACACGACAATCTTGTGTTGGAAAGAAATGCTATTGTCTCGAG
Ⅴ、印鼠客蚤多肽FS48的基因克隆到pET-17b表达载体:在微量离心管中加入1μg含多肽FS48基因的pMD19-T载体、2μl H缓冲液,0.5μl NdeⅠ和0.5μl XholⅠ限制性内切酶,加入水致使最终体积为20μl;1μg pET-17b质粒采用同样的酶切体系。酶切条件为:37℃反应2小时;将酶切产物分别用1%琼脂糖凝胶电泳,切取目的条带,用DNA纯化试剂盒进行纯化。将纯化的目的片段按照摩尔3:1(多肽基因:pET-17b载体)的比例混合加入到等量的连接酶缓冲液混合物中进行连接。连接调节为16℃反应8小时;将连接产物转化进Takara公司的大肠杆菌DH5α感受态细胞。取适量转化产物涂布至含Amp的LB平板上,经37℃培养16小时形成的单菌落即为含目的片段的阳性克隆进行酶切和测序鉴定。
实施例2印鼠客蚤多肽FS48的大肠杆菌表达
将含印鼠客蚤多肽FS48的基因的表达载体质粒化学转化到BL21(DE3)pLysS大肠杆菌中重组表达,1L LB培养基中接入10ml过夜培养种子菌液,37℃培养3小时,当细菌吸光值OD600达到0.8左右时加入IPTG终浓度为1mM,继续培养3小时,8000rpm 10分钟离心收集菌体,用100ml pH 8.0的25mM Tris-HCl溶液悬浮沉淀,然后离心8000rpm离心10分钟收集菌体,去掉上清,向沉淀中加入40ml pH 8.0的25mM Tris-HCl溶液,4℃超声波破碎细胞,超30s停30s连续4次,然后12000rpm离心30分钟收集上清,用3kDa透析袋透析,在10L pH 6.0的PBS溶液中透析24小时。SDS-PAGE检测蛋白的表达,得到图1,目的条带应该在6kDa附近。
实施例3印鼠客蚤多肽FS48大肠杆菌表达产物的分离纯化
第一步,Hiprep SP阳离子交换色谱柱层析。按上述方法透析印鼠客蚤多肽FS48的大肠杆菌表达产物后14000rpm 4℃离心60min去除沉淀,上清液上样于GE公司的Hiprep SP阳离子交换色谱柱。Hiprep SP阳离子交换色谱柱用20mM Na2HPO4-NaH2PO4 pH 6.0的缓冲液平衡,用含1M NaCl的同样缓冲液从0%至100%的线性梯度洗脱得到图2A,收集如图所示活性峰。
第二步,Sephadex SR-100凝胶过滤柱层析。Hiprep SP阳离子交换色谱柱层析得到的活性峰经3kDa超滤管浓缩后上样于25mM Tris-HCl 0.15M NaCl pH 8.0的溶液平衡的Sephadex SR-100凝胶过滤柱。用同样缓冲液洗脱得到图2B,收集如图所示活性峰。
第三步,Hiprep SP阳离子交换色谱柱层析。Sephadex SR100凝胶过滤得到的活性峰用超滤管浓缩更换为20mM Na2HPO4-NaH2PO4 pH 6.0缓冲液后上样于GE公司的Hiprep SP阳离子交换色谱柱。Hiprep SP阳离子交换色谱柱用20mM Na2HPO4-NaH2PO4 pH 6.0的缓冲液平衡,用含1M NaCl的同样缓冲液从0%至100%的线性梯度洗脱得到图2C,收集如图所示活性峰。
第四步,SephadexTM G75 10/300GL凝胶过滤柱层析。Sephadex SR-100凝胶过滤柱层析得到的活性峰经3kDa超滤管浓缩后上样于生理盐水或PBS平衡的SephadexTM G75 10/300GL凝胶过滤层析柱。用同样缓冲液洗脱得到图2D,收集如图所示活性峰。
第五步,纯化得到的印鼠客蚤多肽FS48电泳后得到图2D中的电泳图,然后切胶用自动氨基酸测序仪测定N末端氨基酸序列。
通过上述方法制备的印鼠客蚤多肽FS48是利用印鼠客蚤唾液腺基因重组表达的一种多肽,分子量为6182.26道尔顿,等电点为8.481,具体序列如SEQ ID NO:1所示。
MISATEYKCKVTGTGDMTIPFGCKDGNDVQGCKKLCQEKCKYTTTRQSCVGKKCYC(SEQ ID NO:1)
实施例4印鼠客蚤多肽FS48注射液制备:
(1)取纯化鉴定的印鼠客蚤多肽FS48纯品经3kDa超滤管浓缩后将缓冲液更换为生理盐水或pH值7~8的PBS缓冲液,采用Lowry氏法测定印鼠客蚤多肽FS48含量,调整浓度为10mg/ml注射液母液,分装、-20℃保存。
(2)向注射液母液中加入终浓度为1%的Triton X-114立即剧烈振荡混匀冰育10min后,20℃孵育10min,10000g室温离心10分钟,于超净工作台内小心吸取上清液至无热源器皿中,经鲎试剂检测内毒素小于10EU/mL后将注射液母液调整浓度回10mg/ml待用。
(3)去内毒素的10mg/ml注射液母液用生理盐水或pH值7~8的PBS缓冲稀释成0.5mg/ml的使用液后过滤除菌,-20℃保存分装待用。
印鼠客蚤多肽FS48注射液的药理学效果证实:
实施例5印鼠客蚤多肽FS48注射液对钾离子通道的抑制作用
1)膜片钳分析印鼠客蚤多肽FS48注射液对大鼠背根神经元的作用:挑选出生4周左右、体重约为140~200g的SD大鼠,麻醉后断颈处死,迅速挑出脊椎并将其剪成2~4段,将椎管沿与肋骨垂直的方向剪开,然后在盛有少量培养液的烧杯中浸泡椎管;仔细撕破附在椎管内壁上的无色黏膜,暴露椎管和肋骨交汇处的背根神经纤维。在胸椎和腰椎部分,挑选10~15个较好的背根神经节放入装有2mL临时培养液的培养皿中。分离出神经节以及神经纤维后,用维娜斯剪切除神经节外的絮状物和轴索,放入盛有约0.5mL临时培养液的培养皿中。倒去临时培养液,用维娜斯剪将分离的神经节剪碎。然后将剪碎后神经细胞转入含有15mL消化液的指管中,在34℃下、110rpm的环境中用消化液酶解20~30min。酶解期间每隔8~10min取出指管吹打细胞以防止细胞成团;酶解终止后向消化液中加入胰蛋白酶抑制剂,终止酶解反应。将酶解后的溶液转入离心管中离心(1000rpm、2-5min),去除上清。用含10%小牛血清的培养液重悬细胞,重悬后的细胞分成3~4皿,每皿加入2mL培养液,放入37℃恒温培养箱(5%CO2、95%空气)中,培养3~4小时后用于记录电流。
实验前必须将细胞外液置于室温下平衡后再更换培养皿内的培养液,以防止溶液温度的剧烈变化。更换溶液时要防止细胞从培养皿底部脱落。倒置显微镜下选择细胞膜较为光滑、细胞质均匀的细胞,在室温20~25℃条件下进行膜片钳实验。选用100μl硼硅酸盐玻璃毛细管为玻璃电极材料,玻璃电极在拉制仪(PC-10,Narishige)上经两步拉制而成,玻璃电极热抛光后电极尖端口径为1.5~3.0μm,拉制完成后在玻璃电极内灌细胞内液。玻璃电极初始电阻为1.5~2.5MΩ比较好。待电极与细胞膜之间形成高阻抗的京欧封接后,补电极快电容。然后将细胞钳制在-60mV,给予一短而有力的负压,将钳制在电极中的细胞膜迅速打破,再补偿细胞慢电容。形成全细胞记录模式后将细胞钳制为-80mV,细胞稳定4~6min开始记录电流。Ca2+、Na+和K+电流被记录用全细胞膜片钳模式,用EPC-10放大器(HEKA,Lambrecht/Pfalz,德国),P/4实验方法被运用减去线性电容和漏电电流。实验数据用(version 8.31;HEKA)脉冲程序获取和用Sigmaplot(Sigma)分析。结果如图3A所示,印鼠客蚤多肽FS48能抑制大鼠背根神经元钾离子通道。
2)膜片钳分析印鼠客蚤多肽FS48注射液对表达离子通道细胞系的作用:脂质体将离子通道质粒和绿色荧光蛋白质粒共转染进HEK 293细胞,细胞用含10%胎牛血清的DMEM中在5%CO2,37℃条件下培养。将1.7mm软质玻璃毛坯(VWR,West Chester,PA)用P-97拉制仪拉制抛光后形成电极尖端口径为1.5~3.0μm玻璃电极。膜片钳分析多肽对Kv钾通道作用的离子通道内外液分别为:140mM KF,1mM EGTA,5mM ATP-Na,10mM HEPES,4mMMgCl2,pH7.3溶液和137mM NaCl,5.9mM KCl,1.2mM MgCl2,2.2mM CaCl2,10mM HEPES,14mM glucosepH 7.3溶液。将附有细胞的载玻片加入到灌流槽中,放置在倒置显微镜工作台上,用含100%饱和氧细胞外液灌流装置灌流,选取纹理清晰,表面无颗粒,无收缩的细胞作为实验对象。钳制电极充灌细胞内液后用注射器加入少许正压,使得电极尖端形成一向外凸的液面以便有助于电极入水时保持干净。在液面上选择干净处让电极入水,玻璃电极初始电阻为1.5~2.5MΩ比较好。待电极与细胞膜之间形成高阻抗的京欧封接后,补电极快电容。然后将细胞钳制在-60mV,给予一短而有力的负压,将钳制在电极中的细胞膜迅速打破,再补偿细胞慢电容。形成全细胞记录模式后将细胞钳制为-80mV,细胞稳定4~6min开始记录电流。系统电阻(Rs)在实验过程中始终保持在5~10MΩ的范围之内最好能维持不变,系统串联电阻(Rseries compensation)补偿一般在30~70%之间,实验过程中系统电阻始终保持在5~10MΩ的范围之内。记录电流信号经EPC-10膜片钳放大器以3kHz和10kHZ双重滤波过滤。线性漏电流和电容电流用P/4程序予以删除。实验数据用(version 8.31;HEKA)脉冲程序获取和用Sigmaplot分析。结果如图3所示,印鼠客蚤多肽FS48能抑制Kv1.1和Kv4.1钾离子通道,对Kv1.1抑制的IC50是72.4nM,不影响Kv1.1的稳态活化。
实施例6印鼠客蚤多肽FS48注射液体内镇痛抗炎效果
实验采用鼠尾热痛、福尔马林致痛、醋酸扭体实验模型。实验小鼠选用出生4周左右的单一性别(雄性),体重18-22g之间,分为实验组(10、50、250nM/Kg)以及空白组。
1)鼠尾热痛实验:小鼠实验前先称量体重,按照体重腹腔注射相应体积的三种浓度药物,半小时后将小鼠装入套管。仪器(泰盟SW-200光热尾痛测试仪)预热数分钟将光强度调至062档,探测方式为自动,将小鼠尾部后三分之一段放到检测孔上,待仪器显示准备就绪后打开强光源,当鼠尾受热甩尾时,仪器自动停止计时,结束实验,记录时间。结果如图4A所示,印鼠客蚤多肽FS48能抑制小鼠的热痛,且呈现剂量依赖关系。
2)福尔马林致痛实验:小鼠实验前先称量体重,按照体重腹腔注射相应体积的三种浓度药物,半小时后给每只小鼠按体重给予右后脚爪皮下1%福尔马林溶液20μL。疼痛记录分为两相,phase I(0-5min),phase II(15-45min),记录该时段小鼠舔爪总时间。结果如图4B所示,印鼠客蚤多肽FS48能抑制福尔马林对小鼠的致痛作用,且呈现剂量依赖关系。
3)醋酸扭体实验:小鼠实验前先称量体重,按照体重腹腔注射相应体积的三种浓度药物,半小时后给每只小鼠按体重给予腹腔注射100μL0.68%醋酸,记录0-30min小鼠扭体次数。扭体判断标准为表现为腹部收内凹、腹前壁紧贴笼底、臀部歪扭和后肢伸张,呈一种特殊姿势。结果如图4C所示,印鼠客蚤多肽FS48能抑制醋酸对小鼠的致痛作用,且呈现剂量依赖关系。
4)脚爪肿胀实验:小鼠实验前先称量体重,按照体重腹腔注射相应体积的三种浓度药物,体积为100μL,半小时后给每只小鼠按体重给予右后爪皮下注射100μL1%角叉菜胶,使用泰盟PV-200足趾容积测量仪记录不同时间段小鼠爪子体积。在注射100μL 1%角叉菜胶4h后将脚爪从跗骨处砍下,称重,在含0.5%的十六基溴化三甲胺PBS,pH6.0溶液中匀浆,然后13000g离心3分钟,上清液被转入96孔板,3,3-二甲氧基联苯胺和1%过氧化氢的溶液中,每个样品重复三个孔。设置髓过氧化物酶阳性对照孔。孵育10min后在酶标仪上测定450nm波长吸光值。髓过氧化物酶的活性单位被定义为在22℃1min内使1μM过氧化氢转化成水为1个活性单位。结果如图5所示,印鼠客蚤多肽FS48能抑制甲叉菜胶诱导的炎症,且抑制了脚掌中炎性标志物髓过氧化物酶的活性。
实施例7印鼠客蚤多肽FS48的抗肿瘤效果
1)划痕实验:高表达Kv1.1亚型钾通道细胞系NCI-H460以每孔3×105个接种到六孔板内,待细胞融合度达到80%左右时,在板底部用记号笔做10个点作为拍照时的坐标,将培养基换成没有FBS的RPI1640,并用10μL枪头划三条痕,轻轻晃动用PBS洗掉漂浮的细胞,加入FS48使得浓度为16μΜ,在倒置显微镜下拍照。观察24小时及48小时后加药组与空白组的差异。数据处理采用ImageJ软件。结果如图6所示,印鼠客蚤多肽FS48抑制NCI-H460迁移。
2)侵袭实验:Transwell内孔加入用无血清培养基悬浮的高表达Kv1.1亚型钾通道细胞系NCI-H460细胞悬液,细胞浓度为3×105个每毫升,每孔加入200μL,外孔为含有10%FBS的全培养基RPI1640 500μL,待细胞在37℃5%CO2培养箱稳定4小时后向内孔加入FS48使得终浓度为16μΜ,培养24小时后,取出内孔,用医用棉签轻轻擦去孔内细胞后放置在4%甲醛内固定30min,用PBS清洗后使用10%结晶紫染液染色15min,显微镜下拍照。再用10%醋酸脱色后测量在570nm下的吸光度。结果如图7所示,印鼠客蚤多肽FS48抑制NCI-H460侵袭。
实施例8印鼠客蚤多肽FS48的免疫调节作用
小鼠被无菌脱颈椎处死,无菌取出脾脏,用RPMI1640培养基去掉表明的血液,然后用1ml无菌注射器使其分散成单个细胞,利用红细胞裂解液裂解去掉红细胞,1500rpm离心5分钟,去掉上清,沉淀细胞用含10%小牛血清的RPMI1640重悬,接种105个/孔细胞到96孔细胞培养板,然后加入不同浓度的FS48无菌蛋白和0.75μg/Ml Con A设置空白细胞对照,在5%CO2,37℃条件下培养30min,加入10%浓度的台盼蓝染色液继续培养48h,细胞板测定570nm吸光值。细胞上清中细胞因子的含量采用BD公司的OptEIATM set MOUSE TNF,IL-2,INF捕获ELISA试剂盒检测。结果如图8所示,印鼠客蚤多肽FS48能抑制小鼠脾细胞增殖,抑制IL-2和INF-γ释放,促进TNF-α释放。
SEQUENCE LISTING
<110> 南方医科大学
<120> 印鼠客蚤多肽及其基因和应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 56
<212> PRT
<213> Xenopsylla cheopis
<400> 1
Met Ile Ser Ala Thr Glu Tyr Lys Cys Lys Val Thr Gly Thr Gly Asp
1 5 10 15
Met Thr Ile Pro Phe Gly Cys Lys Asp Gly Asn Asp Val Gln Gly Cys
20 25 30
Lys Lys Leu Cys Gln Glu Lys Cys Lys Tyr Thr Thr Thr Arg Gln Ser
35 40 45
Cys Val Gly Lys Lys Cys Tyr Cys
50 55
<210> 2
<211> 32
<212> DNA
<213> 人工序列
<400> 2
ggcatatgat aagtgcaact gagtataaat gc 32
<210> 3
<211> 24
<212> DNA
<213> 人工序列
<400> 3
ctcgagacaa tagcatttct ttcc 24
<210> 4
<211> 177
<212> DNA
<213> 人工序列
<400> 4
catatgataa gtgcaactga gtataaatgc aaagtaacag gaacaggtga tatgacaata 60
ccatttggat gtaaagatgg caatgatgtt caaggatgta aaaaactttg tcaggagaaa 120
tgtaaataca cgacaacacg acaatcttgt gttggaaaga aatgctattg tctcgag 177
Claims (4)
1.氨基酸序列如SEQ ID NO.1所示的印鼠客蚤多肽FS48或者包含上述所述的多肽的药物组合物在制备抗肿瘤的药物中的用途。
2.氨基酸序列如SEQ ID NO.1所示的印鼠客蚤多肽FS48或者包含上述所述的多肽的药物组合物在制备镇痛和/或抗炎的药物中的用途。
3.氨基酸序列如SEQ ID NO.1所示的印鼠客蚤多肽FS48或者包含上述所述的多肽的药物组合物在制备抑制钾离子通道Kv1.1的药物中的用途。
4.根据权利要求1-3任一所述的用途,其特征在于,所述印鼠客蚤多肽FS48的制备方法包括以下步骤:
(1)提取印鼠客蚤唾液腺总mRNA,并采用SEQ ID NO:2所示的上游引物和SEQ ID NO:3所示的下游引物扩增序列如SEQ ID NO:4所示的印鼠客蚤的基因,然后先将该基因克隆到pET-17b表达载体中,再转化到BL21(DE3)pLysS大肠杆菌中重组表达,并收集目的细胞超声上清;
(2)将所收集的上清透析后依次上Hiprep SP阳离子交换色谱柱、Sephadex SR-100凝胶过滤柱、Hiprep SP阳离子交换色谱柱和SephadexTM G75 10/300GL凝胶过滤柱层析,得到如SEQ ID NO:1所示的印鼠客蚤唾液腺基因重组表达蛋白,即印鼠客蚤多肽FS48。
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