褐黄血蜱卵蛋白的鉴定及在卵孵化过程中的量变

doi:10.16431/j.cnki.1671-7236.2022.11.032

摘要/Abstract

摘要： 【目的】探明褐黄血蜱卵蛋白成分及其在胚胎发育过程中的作用，为筛选抗蜱生殖疫苗分子奠定基础。【方法】提取褐黄血蜱孵化0、7、14、21 d的卵蛋白，以过滤器辅助样品制备（filter-aided sample preparation，FASP）法酶解卵蛋白，液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry，LC-MS/MS）分离、检测酶解特异性肽段，搜索该蜱中肠转录组文库鉴定卵原生质中的蛋白，登录NCBI对各蛋白进行注释；以Maxquant计算各蛋白的iBAQ （intensity-based absolute quantification）和非标记定量（Label-free quantification，LFQ），分析各蛋白在不同孵化时间的相对丰度及其增减，并对高可信蛋白序列进行GO功能富集分析。【结果】本研究共检出特异性肽段1 044条，由此鉴定多肽291种，其中高可信的（unique peptides≥2）112种，但仅39种有相关文献。基于文献，按照功能将39种蛋白归类于酶、酶抑制剂、转运蛋白、热休克蛋白、细胞骨架蛋白及未知蛋白6类；酶、酶抑制剂、转运蛋白、热休克蛋白都有家族蛋白共存的情况。定量分析显示，在新产褐黄血蜱卵中，丰度最高的是Contig14782，其次为Contig6575。至孵化结束时，在20种高丰度蛋白中，1种（Contig2242）显著升高（P<0.05），14种显著下降（P<0.05），5种增减不显著。GO功能富集分析表明，本次鉴定的高可信蛋白主要定位于胞外区域，参与免疫调节过程，发挥结合蛋白质和RNA的作用。【结论】褐黄血蜱卵蛋白成分复杂、种类众多，包括酶、酶抑制剂、转运蛋白、热休克蛋白、细胞骨架蛋白等，其中酶是最多的一类。在孵化期间，绝大多数蛋白酶和酶抑制剂丰度显著下降。

关键词: 褐黄血蜱; 卵蛋白; 液相色谱-串联质谱; 定量分析

Abstract: 【Objective】 The objective of this study was to investigate the protein components of Haemaphysalis flava egg and their roles in embryonic development, so as to lay a foundation for the screening vaccine antigen of anti-tick reproduction.【Method】 The egg proteins of Haemaphysalis flava incubated for 0, 7, 14 and 21 days were extracted and digested by the FASP method.The unique peptides were separated and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.The midgut transcriptome library of Haemaphysalis flava was searched to identify proteins and annotated with NCBI.The iBAQ and LFQ of each protein were calculated by Maxquant, and the abundance of each protein at different incubation time was analyzed.【Result】 1 044 unique peptides were detected and 291 proteins were identified, 112 of which were high confidence (unique peptides ≥ 2), only 39 existing relevant literatures. The 39 proteins had been studied belong to six groups, including enzyme, protease inhibitor, transporter, heat shock protein, cytoskeleton protein, and unknown protein.Enzymes, protease inhibitors, transporters, and heat shock proteins all coexisted with family proteins.Quantitative analysis showed that the highest abundance was Contig14782, followed by Contig6575 in fresh eggs of Haemaphysalis flava.By the end of incubation, among the 20 highly abundant proteins, only Contig2242 was significantly increased (P<0.05), 14 were significantly decreased (P<0.05), and 5 were not significantly changed.GO functional enrichment analysis showed that the high confidence proteins identified in this work were mainly localized in the extracellular space, which was involved in immunoregulatory processes, and the molecular functions were correlated with protein binding, and RNA binding.【Conclusion】 Haemaphysalis flava egg protein composition was complex and numerous, including enzyme, protease inhibitor, transporter, heat shock protein, cytoskeleton protein, and unknown protein, of which enzyme was the most one.During incubation, most enzymes and protease inhibitors decreased significantly.

Key words: Haemaphysalis flava; egg proteins; LC-MS/MS; quantitative analysis

中图分类号:

S852.74^＋6

徐义葳, 李丹, 程天印. 褐黄血蜱卵蛋白的鉴定及在卵孵化过程中的量变[J]. 中国畜牧兽医, 2022, 49(11): 4410-4419.

XU Yiwei, LI Dan, CHENG Tianyin. Identification of Haemaphysalis flava Egg Proteins and Their Expression Changes During Embryonic Development[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4410-4419.

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