捻转血矛线虫Hc-FAR-4蛋白的表达特性及其与配体结合能力分析

doi:10.3864/j.issn.0578-1752.2019.17.013

摘要/Abstract

摘要：

【目的】捻转血矛线虫(Haemonchus contortus)寄生在牛羊等反刍动物的皱胃中,引起的捻转血矛线虫病在我国呈全国性流行。文章通过研究脂肪酸与视黄醇结合相关蛋白Hc-FAR-4的表达特性及配体结合能力,以了解其在捻转血矛线虫生长、发育、繁殖过程中的作用。【方法】对Hc-far-4进行克隆、并且构建原核表达载体pET-30a-Hc-far-4,pET-30a-Hc-far-4重组质粒经过PCR与双酶切鉴定准确无误之后转化到E. coli BL21感受态细胞中,用0.1 mmol·L ^-1IPTG(isopropyl-β-d-thiogalactoside)进行重组蛋白的诱导表达。收集重组蛋白rHc-FAR-4,利用荧光分析法,研究rHc-FAR-4蛋白与DAUDA、视黄醇、油酸的结合能力。配体结合试验是依据荧光物质retinol与脂肪酸类似物DAUDA在极性和非极性溶液中,在某一波长的激发光激发下,所发出的荧光光谱随之变化的特性,判断rHc-FAR-4蛋白是否具有与DAUDA、retinol结合的能力。再在体系中加入非荧光长链脂肪酸油酸,根据荧光光谱的变化情况判定油酸能否分别与DAUDA、retinol竞争目的蛋白的脂肪酸结合位点,间接说明目的蛋白能否与非荧光脂肪酸油酸结合。同时制备鼠源多克隆抗体,利用ELISA技术检验免疫后小鼠的抗体效价,抗体效价合适即可收集小鼠血清。收集的血清用于免疫组织荧光（IHF）试验,探究Hc-FAR-4的表达部位,从而推测其功能。该试验过程为：对捻转血矛线虫进行石蜡包埋,石蜡切片,抗原修复,3%的BSA 于4℃封闭过夜,自制鼠源多克隆抗体作为一抗孵育1 h; Alexa Fluor? 488 nm羊抗鼠IgG作为二抗避光孵育1 h,DAPI染色30 min,激光共聚焦显微镜检查染色情况。利用荧光定量PCR技术分析Hc-far-4在捻转血矛线虫各个主要阶段的表达特性。【结果】成功克隆目的基因Hc-far-4,测序结果与Sanger数据库中捻转血矛线虫far-4基因序列（>HCISE00908800.t1）相似度为 99.9%;重组质粒 pET-30a-Hc-far-4在E. coli BL21中成功表达,在诱导8 h后达到峰值。经过ELISA检测,结果显示制备的鼠源多克隆抗体效价达到1﹕1 024 000—1﹕2 048 000,可用于后续试验。Western Blot鉴定结果显示rHc-FAR-4重组蛋白带有His标签,并且条带大小为25 kD,结果符合预期。制备的鼠源多克隆抗体经过Western Blot鉴定,能够与天然Hc-FAR-4 蛋白结合,说明该抗体可用于IHF试验。配体结合试验结果表明 rHc-FAR-4蛋白具有结合脂肪酸与视黄醇的能力。荧光定量PCR分析表明,Hc-far-4在四期幼虫中的转录水平最高;IHF试验表明,Hc-FAR-4主要表达在捻转血矛线虫的肠壁、性腺中。综上推测Hc-FAR-4蛋白主要在寄生生活阶段参与了转运脂肪酸与视黄醇,为捻转血矛线虫的生长发育与生殖提供营养物质。【结论】捻转血矛线虫 FAR-4 蛋白能够结合脂肪酸类似物DAUDA和视黄醇,但是与油酸的结合能力较弱,主要表达在肠壁,在性腺、角皮中也有少量表达,其基因在进行营寄生生活阶段时表达量达到峰值。

关键词: 捻转血矛线虫, Hc-far-4, 表达特性, 配体结合能力, 脂肪酸

Abstract:

【Objective】 Haemonchus contortus is a parasite mainly settled in the abomasum mucosa of small ruminants. The disease caused by H. contortus is a national epidemic in China. In this paper, the expression pattern and ligand binding ability of Hc-FAR-4 were studied to understand its role in the growth, development and reproduction of H. contortus.【Method】 The prokaryotic expression vector pET-30a-Hc-far-4 was constructed, which was identified by PCR and enzyme digestion identification, and then, the recombinant plasmid was transformed into E. coli BL21. The recombinant protein was induced by 0.1 mmol·L ^-1 IPTG (isopropyl-β-d-thiogalactoside). The recombinant protein rHc-FAR-4 was collected and was identified by Western Blot. Fluorescence analysis method, and then, which was used to study the binding ability of Hc-FAR-4 protein with DAUDA, retinol, and oleic acid. The IHF experiment was based on the fluorescent material retinol and fatty acid analogues DAUDA in polar or nonpolar solution, and their excitation spectra characteristics would change when they were excitated by a certain wavelength of exciting light. Using fluorescence analysis method, we could estimate whether rHc-FAR-4 protein contained the ability to bind with DAUDA and retinol by the change of the excitated spectra. When non-fluorescent oleic acid was added into the system, it would compete with DAUDA and retinol to bind with the bind site of rHc-FAR-4 protein, and which would make excitated spectral change, too. According to the changes, we could indirectly judge whether rHc-FAR-4 protein could bind with non-fluorescent oleic acid. At the same time, polyclonal antibodies were prepared by using rHc-FAR-4 protein to immunize mice, and the antibody titer of the immunized mice was tested by ELISA. The serum of the immunized mice would be collected if the antibody titer was appropriate. The collected serum was used in the immunofluorescence (IHF) experiment to explore the expression site of Hc-FAR-4 protein to speculate its function in H. contortus. The IHF experiment process was as follow: The H. contortus was embedded in paraffin; the paraffin was cut into slices; Antigen was repaired; the slices was incubated by 3% BSA at 4℃ for one night; slices was incubated by anti-Hc- FAR-4 mouse antibody and Alexa Fluor? 488 nm goat anti-mouse IgG antibody for 1 h, and which was done as primary and secondary antibody, respectively; after staining by DAPI, the slices were observed by confocal microscopy. What ^’s more, the qPCR technology was used to analyze the expression characteristics of Hc-far-4 in H. contortus.【Result】 The target gene Hc-far-4 was successfully cloned. The recombinant plasmid pET-30a-Hc-far-4 was successfully expressed in E. coli BL21. The recombinant plasmid pET-30a-Hc-far-4 was successfully expressed in E. coli BL21, and the expression level of rHc-FAR-4 protein reached peak after induction for 8h. The result of ELISA showed that the titer of mouse polyclonal antibody was 1:1 024 000—1:2 048 000, which could be used in the following experiments. Western Blot result showed that the rHc-FAR-4 protein contained His tag and the band size was 25 kD, which was consistent with the prediction. The mouse polyclonal antibody was identified by Western Blot and could bind to natural Hc-FAR-4 protein, indicating that the antibody could be used in IHF experiment. The results of ligand binding experiments of rHc-FAR-4 showed that rHc-FAR-4 could bind to fatty acids and retinol. The qPCR analysis showed that Hc-far-4 reached the highest transcription level in the fourth stage larvae. Immunohistofluorescence assay showed that Hc-FAR-4 was mainly expressed in the intestinal wall and gland of H. contortus. To sum up, it could be speculated that Hc-FAR-4 protein might be involved in the transport of fatty acids and retinol, and provided nutrients for H. contortus to ensure its normal growth, development and reproduction; what ^,s more, Hc-FAR-4 protein maybe also involved in the process of modifying host tissues to enable the parasite to escape the immunity of host.【Conclusion】 rHc-FAR-4 could bind to DAUDA and retinol, and which was mainly expressed in the intestinal wall, cuticle and gonad. The transcription level of Hc-far-4 reached the peak in the fourth stage larvae.

Key words: Haemonchus contortus, Hc-far-4, expression pattern, ligand binding capacity, fatty acids

韦海典, 陈学秋, 黄艳, 时恒枝, 周静茹, 吴飞, 杜爱芳, 杨怡. 捻转血矛线虫Hc-FAR-4蛋白的表达特性及其与配体结合能力分析[J]. 中国农业科学, 2019, 52(17): 3059-3068.

WEI HaiDian, CHEN XueQiu, HUANG Yan, SHI HengZhi, ZHOU JingRu, WU Fei, DU AiFang, YANG Yi. The Expression Pattern and Ligand Binding Ability of Hc-FAR-4 Protein of Haemonchus contortus[J]. Scientia Agricultura Sinica, 2019, 52(17): 3059-3068.

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参考文献 31

[1]	WANG C R, QIU J H, ZHU X Q, HAN X H, NI H B, ZHAO J P, ZHOU Q M, ZHANG H W, LUN Z R . Survey of helminths in adult sheep in Heilongjiang Province, People's Republic of China. Veterinary Parasitology, 2006,140:378-382.
[2]	BESIER R B, KAHN L P, SARGISON N D, WYK J A V . The pathophysiology, ecology and epidemiology of Haemonchus contortus infection in small ruminants. Advances in Parasitology, 2016,93:95-143.
[3]	沈效平 . 湖南山羊捻转血矛线虫感染调查及 rDNA ITS遗传差异分析[D]. 长沙: 湖南农业大学, 2013.
	SHEN X P . Investigating prevalence and researching genetic differences of rDNA ITS of Haemonchus contortus from goat in Hunan[D]. Changsha: Hunan Agricultural University, 2013. (in Chinese)
[4]	解晓钰 . 博州地区羊消化道寄生虫感染调查及冬季驱虫试验研究[D]. 乌鲁木齐: 新疆农业大学, 2009.
	XIE X Y . Investigation on the infection of digestive parasites in sheep in Bo-zhou area and winter expel parasites experiment study[D]. Wurumuqi: Xinjiang Agricultural University, 2009. (in Chinese)
[5]	吴震洋, 冉辉, 朱天钧, 李丽 . 羊捻转血矛线虫病防控研究进展. 安徽农学通报, 2017(8):123-124.
	WU Z Y, RAN H, ZHU T J, LI L . Advances in the prevention and control ofHaemonchus contortus in sheep. Anhui Agricultural Science Bulletin, 2017(8):123-124. (in Chinese)
[6]	KOTZE A C, PRICHARD R K . Chapter nine-anthelmintic resistance in Haemonchus contortus: history, mechanisms and diagnosis. Advances in Parasitology, 2016,93:397-428.
[7]	ALBUQUERQUE A C A D, BASSETTO C C, ALMEIDA F A D, ALESSANDRO F T A . Development of Haemonchus contortus resistance in sheep under suppressive or targeted selective treatment with monepantel.Veterinary Parasitology, 2017, 246:112-117.
[8]	LEITE D S, MONTEIRO J, RIBEIRO C, MACEDO F, FILHO A, ANDRE P . High levels of benzimidazole resistance and beta-tubulin isotype1 SNPF167Y in Haemonchus contortus populations from Ceara State, Brazil. Small Ruminant Research, 2016,146:48-52. doi: 10.1016/j.smallrumres.2016.11.023.
[9]	KEARNEY P E, MURRAY P J, HOY J M, HOHENHAUS M, KOTZE A . The ‘Toolbox’ of strategies for managing Haemonchus contortus in goats: What’s in and what’s out. Veterinary Parasitology, 2016, 220:93-107.
[10]	CAMPBELL B E, BOAG P R, HOFMANN A, CANTACESSI C, WANG C K, TAYLOR P . Atypical (rio) protein kinases from Haemonchus contortus promise as new targets for nematocidal drugs. Biotechnology Advances, 2011, 29(3), 338-350.
[11]	郑秀平, 丁豪杰, 郭筱璐, 杨怡, 黄艳, 陈学秋, 周前进, 杜爱芳 . 捻转血矛线虫Hc-daf-22基因的原核表达及重组蛋白酶活性测定. 中国农业科学, 2017,50(8):1535-1542. doi: 10.3864/j.issn.0578-1752.2017.08.017
	ZHENG X P, DING H J, GUO X L, YANG Y, HUANG Y, CHEN X Q, ZHOU Q J, DU A F . Characteristics of Hc-daf-22 gene from Haemonchus contortus crokaryotic expression and its enzymatic activity. Scientia Agricultura Sinica, 2017,50(8):1535-1542. (in Chinese) doi: 10.3864/j.issn.0578-1752.2017.08.017
[12]	KUANG L, COLGRAVE M L, BAGNALL N H, KNOX M R, QIAN M, WIJFFELS G . The complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach. Molecular and Biochemical Parasitology, 2009,168(1):84-94.
[13]	TREE T I M, GILLESPIE A J, SHEPLEY K J, BLAXTER M L, TUAN R S, BRADLEY J E . Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus, with homologues in other filarial nematodes and Caenorhabditis elegans. Molecular and Biochemical Parasitology, 1995,69(2):185-195.
[14]	KENNEDY M W, GARSIDE L H, GOODRICK L E, MCDERMOTT L, BRASS A, PRICE N C, KELLY S M, COOPER A, BRADLEY J E . The Ov20 protein of the parasitic nematode Onchocerca volvulus: A structurally novel class of small helix-rich retinol-binding proteins. Journal of Biological Chemistry, 1997,272(47):29442-294488.
[15]	BASAVARAJU S V, ZHAN B, KENNEDY M W, LIU Y, HAWDON J, HOTEZ P J . Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation. Molecular and Biochemical Parasitology, 2003,126(1):63-71
[16]	GAROFALO A . The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties, structural characteristics, and developmental regulation. Journal of Biological Chemistry, 2003a,278(10):8065-8074.
[17]	FAIRFAX K C, VERMEIRE J J, HARRISON L M, BUNGIRO R D, GRANT W, HUSAIN S Z, CAPPELLO M . Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum. International Journal for Parasitology, 2009, 39(14):1561-1571.
[18]	WANG Y, LUO Z, SHI X, LUO Z P, SHI X L, WANG H, NIE L, HUANG M D . A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin. Protein Science A Publication of the Protein Society, 2011,20(12):2095-2101.
[19]	FLORENCIA R B, GABRIELSEN M, FRANCHINI G R . Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus. Biochemical Journal, 2015,471(3):403-414.
[20]	ZHANG L, MOU L, CHEN X, YANG Y, HU M, LI X R, SUO X, ZHU X Q, DU A F . Identification and preliminary characterization of Hc-clec-160, a novel C-type lectin domain-containing gene of the strongylid nematode Haemonchus contortus. Parasites & Vectors, 2018,11(1):430.
[21]	梁忠泉, 袁昌隆, 张宝贺 . 微小组织石蜡切片制作体会. 中国组织化学与细胞化学杂志, 2018(04): 368-373..
	LIANG Z Q, YUAN C L, ZHANG B H . Experience in making paraffin sections of microtissues. Chinese Journal of Histochemistry and Cytochemistry, 2018(04): 368-373.. (in Chinese)
[22]	刘慧军, 杨海明, 胥蕾, 张李荣, 王志跃 . 畜禽肠道组织H. E. 染色石蜡切片制作要点及关键技术. 黑龙江畜牧兽医, 2018(15):206-210.
	LIU H J, YANG H M, XU L, ZHANG L R, WANG Z Y . Key points and key techniques for the preparation of he. staining paraffin sections of animal and poultry intestinal tissues.Heilongjiang Animal Husbandry and Veterinary Science, 2018(15):206-210. (in Chinese)
[23]	范瑾瑾, 骆宁, 彭文兴, 毛海萍 . 石蜡切片多重免疫荧光染色优化条件探讨. 中山大学学报(医学版), 2018,39(05):675-681.
	FAN J J, LUO N, PENG W X, MAO H P . Discussion on optimal conditions for multiple immunofluorescence staining of paraffin sections. Journal of Sun Yat-sen University(medical edition), 2008,39(05):675-681. (in Chinese)
[24]	阳毅敏, 潘灵韬, 庄浩瀚, 孙洪超, 杨怡, 陈学秋, 杜爱芳 . 微小隐孢子虫黏蛋白CGD5_2060原核表达及其黏附功能. 浙江大学学报(农业与生命科学版), 2018,44(2):230-236.
	YANG Y M, PAN L T, ZHUANG H H, SUN H C, YANG Y, CHEN X Q, DU A F . Prokaryotic expression of Cryptosporidium parvum mucin CGD5_2060 and its role in adhesion. Journal of Zhejiang University (Agric. & Life Sci. ), 2018,44(2):230-236. (in Chinese)
[25]	罗丽莲 . 小麦孢囊线虫脂肪酸和视黄醇结合蛋白基因的克隆及表达分析[D]. 武汉: 华中农业大学, 2015.
	LUO L L . Cloning and expression analysis of fatty acid and retinol-binding protein genes of Triticum sporangium nematode[D]. Wuhan: Huazhong Agricultural University, 2015. (in Chinese)
[26]	KENNEDY M W . The polyprotein lipid binding proteins of nematodes. Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology, 2000, 1476(2):149-164.
[27]	RATHORE D K, SUCHITRA S, SAINI M . Identification of a 66kDa Haemonchus contortus excretory/secretory antigen that inhibits host monocytes. Veterinary Parasitology, 2006, 138(3):291-300.
[28]	ANBU K A, JOSHI P . Identification of a 55 kDa Haemonchus contortus excretory/secretory glycoprotein as a neutrophil inhibitory factor. Parasite Immunology, 2010, 30(1):23-30.
[29]	DING H, SHI H, YU S, GUO X L, ZHENG X P, CHEN X Q, ZHOU Q J, YANG Y, DU A F . Characterization and function analysis of a novel gene, Hc-maoc-1, in the parasitic nematode Haemonochus contortus. Parasites & Vectors, 2017,10(1):67.
[30]	郭晓璐 . Hc-dhs-28 基因参与捻转血矛线虫发育过程调控机制[D]. 杭州: 浙江大学, 2017.
	GUO X L . The regulatory mechanism of hc-dhs-28 gene involved in the development process of Haemonochus contortus[D] Hangzhou: Zhejiang University, 2017. (in Chinese)
[31]	张玲 . Hc-clec-160 基因克隆及其在捻转血矛线虫生长发育和繁殖过程中的调节作用[D]. 杭州: 浙江大学, 2018.
	ZHANG L . Cloning of Hc-clec-160 gene and its regulatory role in the growth, development and reproduction of Haemonochus contortus[D]. Hangzhou: Zhejiang University, 2018. (in Chinese)