蜱是一类以吸血为生的体外寄生虫，其生活史包括卵、幼蜱、若蜱和成蜱4个阶段。在卵期，受精卵经过卵裂、囊胚、原肠胚、分节等过程发育成幼蜱，并破壳而出，其间发生的反应众多而有序、参与的物质复杂而有定，尚能探明对了解蜱类胚胎发育规律，创新控蜱策略具有重要意义。
人们很早就已开始了蜱卵原生质组成和胚胎发育过程的研究。1943年，Wigglesworth[1]首先关注蜱卵颜色与血红素的关系。随后，Bremner[2]证实了蜱卵中的色素取决于球蛋白与血红素结合而成的复合物。迄今为止，人们已经从安氏矩头蜱[3]、毛白钝缘蜱[4]、变异矩头蜱[5-6]、肩突硬蜱[7]、微小扇头蜱[8]、长角血蜱[9]卵中分离鉴定了多种卵黄蛋白，以及催化卵黄蛋白和血红蛋白分解的微小扇头蜱的heme-binding aspartic proteinase[10]、Boophilus Yolk pro-Cathepsin[11]、vitellin-degrading cysteine endopeptidase[12]、毛白钝缘蜱的cathepsin L-like proteinase[13]。最近，人们又发现了参与糖类代谢的GSK-3[14]、能清除自由基的GST[15]、具抑制作用的serine proteinase inhibitor[16]、kallikrein inhibitor[17]和抑制细菌生长发育的抗菌肽[18]。人们对蜱卵蛋白成分及其相互关系知之尚少，本研究拟以LC/MS/MS法对豪猪血蜱卵原生质中蛋白质成分进行鉴定。
豪猪血蜱广泛分布于温带和亚热带地区，我国鲁、苏、闽、台、粤、琼、湘、鄂、滇、甘等地均有发现[19]，常寄生于野猪、豪猪、猪獾，偶尔也见于狗、水牛、小麂和人类[20]。该蜱携带锥虫[21]、埃立克体[22]、立克次氏体[23]、包柔氏螺旋体、吉氏巴贝斯虫[24]等多种病原微生物，并有人被叮咬后发生无形体病[25]、日本斑点热[26]的报道，具有一定的公共卫生意义。
1 材料与方法 1.1 材料1.1.1 试剂与仪器 氯仿、甲醛、乙腈、甲酸系国药集团化学试剂有限公司产品；胰蛋白酶购于Promega(北京)生物技术有限公司；Q Exactive质谱仪为Thermo Fisher生产；液相色谱仪购于Agilent Technologies。
1.1.2 豪猪血蜱 豪猪血蜱采自湖南省湘西猪獾体表，由湖南农业大学动物医学院程天印教授鉴定。
1.2 方法1.2.1 蜱卵的收集与处理 将饱血雌蜱置于昆虫培养箱，于28 ℃，85%RH下培养。每日定时收集蜱卵。按徐律和程天印[27]的方法去除卵表腊质。
1.2.2 蛋白的提取与消化 取去腊质卵50 mg，加入0.1 mol·L-1 Tris-HCl溶液0.4 mL，研碎；转移至洁净离心管，加入20%SDS和1M DTT各25 μL，混匀，95 ℃孵育3 min；25 ℃下，16 000 g离心10 min，取上清；冻干，备用。
1.2.3 样品处理 取卵蛋白干粉50 g，加入100 mmol·L-1 DTT 50 μL，沸水浴5 min，冷却至室温；加入8 mol·L-1尿素缓冲液200 μL，混匀，转入10 ku超滤离心管，14 000 g离心15 min。加入100的IAA液，混匀，14 000 g离心10 min。尿素缓冲液洗涤2次。加胰蛋白酶消化液40 μL至滤膜上，600 r·min-1振荡1 min，37 ℃ 16~18 h。14 000 g离心滤液10 min，移至C18小柱，过柱脱盐后冷冻干燥。
1.2.4 质谱分析 以0.1%三氟乙酸溶解上述肽段样粉，自动进样器进样，经捕集柱、色谱柱分离后，质谱仪检测离子。检测方式：正离子；采集方法：每次采集10个碎片图谱。
1.2.5 数据分析 将质谱数据和自建的褐黄血蜱卵巢转录组翻译文库(PRJNA600997)、唾液腺转录组翻译文库(PRJNA280697)和中肠转录组翻译文库(PRJNA286260)肽序列导入Mascot2.2，设定搜索参数，搜索特异性肽段及其匹配多肽。
1.2.6 蛋白序列分析 上传各高可信蛋白序列至Uniprot数据库，进行搜库、注释。
2 结果 2.1 卵总蛋白检测采用BCA法测定卵蛋白粗提液浓度，提取液蛋白含量5.26 μg·μL-1。卵蛋白粗提液的SDS-PAGE结果表明，豪猪血蜱卵原生质所含蛋白多在15~170 ku，且分布不均，相对质量近55、65、80、120和170 ku者丰度尤高。
2.2 卵蛋白鉴定以卵蛋白酶解物质谱检测数据搜索卵巢、唾液腺、中肠转录组文库，分别检出特异性肽段359、312、357条，鉴定蛋白108、170、161种，其中高可信的(unique peptides≥2)依次为60、35、45种(图 2，有部分重叠)。基于序列比对，合并由同一mRNA编码的，最终鉴定高可信蛋白103种，但其中36种尚无相关研究资料(表 1)。
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M. 蛋白质相对分子质量标准；1.样品 M. Protein marker; 1. Sample 图 1 豪猪血蜱卵总蛋白提取液SDS-PAGE图 Fig. 1 Proteins extracted from the eggs of Haemaphysalis hystricis |
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OV、SG、MG分别是卵巢转录组文库、唾液腺转录组文库、中肠转录组文库鉴定到的蛋白 OV, SG and MG represent the proteins identified in ovarian transcriptome library, salivary gland transcriptome library and midgut transcriptome library respectively 图 2 卵巢、唾液腺和中肠转录组文库中鉴定到的高可信蛋白及其重叠情况 Fig. 2 Overlap of highly confidence proteins identified in the ovary, salivary glands, and midgut transcriptome libraries |
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表 1 豪猪血蜱卵蛋白功能分类 Table 1 Functional classification of Haemaphysalis hystricis egg proteins |
基于既有文献，按活性或功能对本研究检出的、有文献参考的67种高可信蛋白进行分类，结果见表 1。表 1列出的67种高可信蛋白分别属于酶类、蛋白酶抑制剂、转运蛋白、细胞骨架蛋白、热休克蛋白和免疫相关蛋白等6类，其中酶类最多(25种)，其次是蛋白酶抑制剂(18种)和转运蛋白(11种)。
2.3 103种高可信蛋白生物信息学分析借助Omics Bean对高可信蛋白进行GO分析，结果表明(图 3)，本次鉴定的高可信蛋白多为细胞外蛋白，参与免疫调节，并且在酶活性调节方面发挥作用。
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图 3 豪猪血蜱卵103种高可信蛋白GO分析结果 Fig. 3 GO analysis of 103 highly confident proteins in Haemaphysalis hystricis eggs |
由于目前尚无豪猪血蜱的基因或蛋白文库，故本研究便基于笔者构建的其近缘蜱种(褐黄血蜱)的卵巢、唾液腺和中肠转录组文库进行其卵蛋白成分鉴定。与笔者所做的褐黄血蜱卵蛋白鉴定结果一致，暗示这一做法可行、可靠。在分析过程中，发现有些蛋白可从不止一个文库搜出，为此，我们首先借助序列比对厘清它们的关系，然后进行取舍或拼接，如SG27993长149 aa，MG1301长373 aa，两者相同部分138 aa，对其进行拼接，然后搜库、注释；MG4254长316 aa、OVN53697长673 aa，MG4254包含于OVN53697，故舍弃MG4254，以卵巢序列OVN53697搜库、注释。
从本次鉴定的高可信蛋白看，无论是酶，还是酶抑制剂和转运蛋白都存在同一家族多个成员并存的情况，笔者称之为家族性。在卵原生质蛋白中比较突出的是Cathepsin、Aspartic protease、Serpins 9、Kunitz、Vitellogenin、Microtubule associated complex、HSP70等。同家族蛋白在胚胎发育过程中如何协调、互补地发挥作用尚不得而知。比对显示，同家族蛋白间的一级结构极其相似，如OVN53099和MG241(Cathepsin L-like cysteine protein)对应部分的相似性达93.16%。
组织蛋白酶类是酶类最多的，且主要是天冬氨酸蛋白酶和半胱氨酸蛋白酶两类，其中前者14种，后者7种。业已证明，Cathepsin D、Heme-binding aspartic peptidase、Cathepsin L-like cysteine protein以及Vitellin-degrading cysteine endopeptidase均可催化卵黄蛋白和血红蛋白水解[12-13, 28-30], 但活性有所差异，如微小扇头蜱的Cathepsin D对卵黄蛋白的活性较高[28]，Vitellin-degrading cysteine endopeptidase对血红蛋白的活性更高[12]。
在蜱卵原生质中还存在大量的蛋白酶抑制剂，其中Serpin类数量最多，其次是Cystatin。人们已从长角血蜱、篦子硬蜱等蜱鉴定到数种Serpin[31-35]，大多数serpin既能抑制丝氨酸蛋白酶，进而影响到血红蛋白的降解[36]，又可抑制糜蛋白酶、弹性蛋白酶和木瓜蛋白酶样半胱氨酸蛋白酶等，起到调节宿主免疫系统的作用[37-38]。Waxman等[39]证实，蜱类的Serpin具有抗凝活性，但机制不尽相同，如毛白钝缘蜱的serpin抑制Xa因子，长角血蜱的serpin 2抑制凝血酶活性[40]。Cystatin是半胱氨酸蛋白酶抑制剂，可抑制Cathepsin L-like cysteine protein[41]以及Vitellin-degrading cysteine endopeptidase[42]等酶的活性，暗示蜱类胚胎发育过程中发生的酶促反应也受控制、调节。
Vg是豪猪血蜱卵中的高丰度成分，种类也较多，如Vitellogenin-1、Vitellogenin-2、Vitellogenin-B以及Vitellogenin。Vg主要由饱血雌蜱的中肠和脂肪体合成，通过淋巴转移至卵巢，然后转化为Vn，作为胚胎发育必须的营养贮存于卵原生质[43]。另外，本次还鉴定了6个脂蛋白和1个脂肪酸结合蛋白。Kluck等[44]证实，微小扇头蜱血淋巴中的一种脂蛋白RmLCP可结合大多数脂肪酸，发挥运输脂类的作用。Campos等[45]认为，脂类和碳水化合物是微小牛蜱胚胎发育的主要能源物质。此次鉴定到的葡萄糖脱氢酶、甘油醛-3-磷酸脱氢酶、转酮醇酶和ATP合酶等4种糖代谢相关酶的存在说明胚胎发育过程中既有糖酵解反应[46]，也有戊糖磷酸途径[47]。
蜱卵原生质蛋白具有开发应用价值。Maria以微小扇头蜱卵源Cathepsin免疫牛，激发了显著的免疫应答[48]。以微小扇头蜱卵源Vitellin-degrading enzyme免疫牛使其获得了保护性[49]。
4 结论经FASP法消化豪猪血蜱卵蛋白提取物，LC/MS/MS法检测结果显示：搜索褐黄血蜱卵巢转录组文库、唾液腺转录组文库和中肠转录组文库，分别检出特异性肽段359、312、357条，鉴定多肽108、170、161条，高可信蛋白103种。豪猪血蜱蜱卵原生质中富含酶类、蛋白酶抑制剂、转运蛋白、细胞骨架蛋白、蛋白质合成与修饰、分泌蛋白以及未鉴定的蛋白等。
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(编辑 白永平)