Primary study on the expression pattern and function of zinc metalloproteinase NAS-31 in <i>Haemonchus contortus</i>

doi:10.3785/j.issn.1008-9209.2020.12.221

Journal of Zhejiang University (Agriculture and Life Sciences)

2021, Vol. 47

Issue (6): 805-814 DOI: 10.3785/j.issn.1008-9209.2020.12.221

Animal sciences & veterinary medicine

Primary study on the expression pattern and function of zinc metalloproteinase NAS-31 in Haemonchus contortus

Yan HUANG(

),Hui ZHANG,Danni TONG,Jingru ZHOU,Fei WU,Xueqiu CHEN,Yi YANG,Guangxu MA,Aifang DU(

)

Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

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Abstract

Haemonchosis is caused by Haemonchus contortus parasitic in the ruminant abomasum. In order to study the function of zinc metalloproteinase NAS-31 gene (Hc-nas-31) in the free-living period (especially L3) of this kind of worm, real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to detect the transcription level of the target gene in different developmental stages of H. contortus. Rapid amplification of cDNA ends (RACE), genome walking and fusion PCR methods were used to amplify Hc-nas-31 gene and the gene structure was analyzed after that. Then, prokaryotic expression plasmid was constructed and transformed into BL21 to express the recombinant protein. A New Zealand white rabbit was immunized with the purified recombinant Hc-NAS-31 (rHc-NAS-31) to prepare polyclonal antibodies, which were used to analyze the expression pattern of Hc-NAS-31 in H. contortus by indirect immunofluorescence assay. The results showed that Hc-nas-31 was transcribed in all stages of the worms, while the transcription level of L3 was the highest. Polyclonal antibody was made successfully and its specific binding with Hc-NAS-31 natural protein was confirmed by Western blotting. Indirect immunofluorescence analysis showed that Hc-NAS-31 protein was mainly expressed in the epithelial syncytia of L3, while in adults, it was distributed in the intestines, gonads, muscle tissues and early eggs. In summary, we confirmed the expression pattern of NAS-31 in H. contortus. This experiment lays the foundation for further research on the biological functions of Hc-nas-31.

Key words： Haemonchus contortus Hc-nas-31 transcription level expression pattern

Received: 22 December 2020 Published: 25 December 2021

CLC:

S 855.9

Corresponding Authors: Aifang DU E-mail: huangyan1990@zju.edu.cn;afdu@zju.edu.cn

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	Yan HUANG
	Hui ZHANG
	Danni TONG
	Jingru ZHOU
	Fei WU
	Xueqiu CHEN
	Yi YANG
	Guangxu MA
	Aifang DU

Cite this article:

Yan HUANG,Hui ZHANG,Danni TONG,Jingru ZHOU,Fei WU,Xueqiu CHEN,Yi YANG,Guangxu MA,Aifang DU. Primary study on the expression pattern and function of zinc metalloproteinase NAS-31 in Haemonchus contortus. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(6): 805-814.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2020.12.221 OR http://www.zjujournals.com/agr/Y2021/V47/I6/805

捻转血矛线虫锌金属蛋白酶NAS-31的表达特性及功能初步研究

捻转血矛线虫病由捻转血矛线虫（Haemonchus contortus）寄生于反刍动物皱胃之中引起。为探究捻转血矛线虫锌金属蛋白酶NAS-31基因（Hc-nas-31）在虫体自由生活时期（特别是3期幼虫）中发挥的功能，本研究通过实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction, qRT-PCR）检测目的基因在捻转血矛线虫各发育阶段的转录水平，利用cDNA末端快速扩增（rapid amplification of cDNA ends, RACE）、基因步移及融合PCR方法扩增并分析其基因结构；构建原核表达载体后转化至BL21感受态细胞中进行诱导表达；将纯化后的蛋白免疫新西兰大白兔以制备多克隆抗体；通过虫体间接免疫荧光分析Hc-NAS-31在捻转血矛线虫体内的表达特性并预测其功能。结果表明：Hc-nas-31在各时期虫体中均有转录，其中在3期幼虫中的转录水平最高；蛋白质印迹结果显示，制备的抗体能特异性结合Hc-NAS-31天然蛋白；间接免疫荧光分析发现， Hc-NAS-31蛋白主要表达于3期幼虫的上皮细胞合胞体内，在成虫中则分布于肠道、生殖腺（性腺）、肌肉组织及早期虫卵中。本实验明确了NAS-31在捻转血矛线虫中的表达特性，为进一步研究Hc-nas-31的生物学功能奠定了基础。

关键词： 捻转血矛线虫, 捻转血矛线虫锌金属蛋白酶NAS-31基因, 转录水平, 表达特性

Table 1 Primer information

Fig. 1 Hc-nas-31 geneamplification and structural schematic diagramA. RACE result; B. 5

´

genome walking amplification; C. 3

´

genome walking amplification; D. Gene structural schematic diagram of Hc-nas-31. M: DNA marker; 1-2: Outer PCR products; 3-4: Inner PCR products; SP1-SP3: PCR products of different primers.

Fig. 2 Amplification of Hc-nas-31 full-length CDS and structural schematic diagram of Hc-NAS-31 proteinM1-M2: DNA markers; 1-2: PCR products; 3: Hc-nas-31 full-length CDS; SP: Signal peptide; CUB: CUB domain; SXC: SXC domain.

Fig. 3 Transcription levels of Hc-nas-31 at different stages of H. contortusSingle asterisk (*) indicates significant differences compared with the female adult (FA) period at the 0.05 probability level; double asterisks (**) indicate highly significant differences compared with the FA period at the 0.01 probability level; and triple asterisks (***) indicate extremely high significant differences compared with the FA period at the 0.001 probability level. L1-L4: Larvae of stage L1-L4; F: Female; M: Male; A: Adult.

Fig. 4 Identification of the recombinant plasmid, induction expression and Western blotting analysis of polyclonal antibodiesA. Identification of the recombinant plasmid (M: DNA marker; 1: Expression vector pET-30a; 2-4: Products of double enzyme digestion); B: Induction expression of pET-30a-Hc-nas-31-BL21 (M: Protein marker; 0-5: Protein products at 0-5 h induction); C. Western blotting analysis of polyclonal antibodies (1: Whole worm proteins of L3; 2: Whole worm proteins of the adult).

Fig. 5 Indirect immunofluorescence analysis of whole worm of L3A. Merge; B. Green fluorescent protein (GFP); C. 4

´

, 6-diamidino-2-phenylindole (DAPI); D. Differential interference contrast (DIC).

Fig. 6 Expression locations of Hc-NAS-31 in male adults of H. contortusPlease see the footnote of Fig. 5 for the details of the abbreviation symbols. A₁-A₄. Negative controls; B₁-B₄, C₁-C₄, D₁-D₄, E₁-E₄. Transverse sections of male adults (g: Gonad; in: Intestine; m: Muscle).

Fig. 7 Expression locations of Hc-NAS-31 in female adults of H. contortusPlease see the footnote of Fig. 5 for the details of the abbreviation symbols. A₁-A₄, B₁-B₄, C₁-C₄. Longitudinal sections of the female adults; D₁-D₄, E₁-E₄, F₁-F₄. Transverse sections of the female adults (in: Intestine; e: Immature egg; g: Gonad).


[1]	GASSER R B, SAMSON-HIMMELSTJERNA G VON. Haemonchus contortus and haemonchosis: past, present and future trends. Advances in Parasitology, 2016,93:1-623. DOI:10.1016/s0065-308x(16)x0004-8 doi: 10.1016/s0065-308x(16)x0004-8

[2]	AHMAD R Z, TIFFARENT R. Pathological aspects of haemonchosis in goats and sheep. WARTAZOA-Indonesian Bulletin of Animal and Veterinary Sciences, 2020,30(2):91-102. DOI:10.14334/wartazoa.v30i2.2185 doi: 10.14334/wartazoa.v30i2.2185

[3]	郭筱璐,杨怡,施宇,等.国内羊胃肠道寄生虫流行病学调查及捻转血矛线虫系统进化分析.中国兽医学报,2018,38(7):1332-1337. DOI:10.16303/j.cnki.1005-4545.2018.07.13 GUO X L, YANG Y, SHI Y, et al. The epidemical survey of sheep gastrointestinal parasites in partial areas of China. Chinese Journal of Veterinary Science, 2018,38(7):1332-1337. (in Chinese with English abstract) doi: 10.16303/j.cnki.1005-4545.2018.07.13

[4]	赵学亮,刘晓磊,苏倩,等.2018年内蒙古察右后旗绵羊捻转血矛线虫流行病学调查及耐药性检测.中国动物检疫,2019,36(5):6-10. DOI:10.3969/j.issn.1005-944X.2019.05.002 ZHAO X L, LIU X L, SU Q, et al. Epidemiological investigation on Haemonchus contortus in sheep in Chayouhouqi of Inner Mongolia and detection of its drug resistance. China Animal Health Inspection, 2019,36(5):6-10. (in Chinese with English abstract) doi: 10.3969/j.issn.1005-944X.2019.05.002

[5]	方变变,李军燕,罗晓平,等.捻转血矛线虫P糖蛋白基因的表达与耐伊维菌素药物相关性.中国兽医学报,2020,40(4):713-718. DOI:10.16303/j.cnki.1005-4545.2020.04.09 FANG B B, LI J Y, LUO X P, et al. Correlation on expression of Pgp and ivermectin resistance in Haemonchus contortus. Chinese Journal of Veterinary Science, 2020,40(4):713-718. (in Chinese with English abstract) doi: 10.16303/j.cnki.1005-4545.2020.04.09

[6]	DE ALBUQUERQUE A C A, BASSETTO C C, DE ALMEIDA F A, et al. Development of Haemonchus contortus resistance in sheep under suppressive or targeted selective treatment with monepantel. Veterinary Parasitology, 2017,246:112-117. DOI:10.1016/j.vetpar.2017.09.010 doi: 10.1016/j.vetpar.2017.09.010

[7]	KOTZE A C, PRICHARD R K. Anthelmintic resistance in Haemonchus contortus: history, mechanisms and diagnosis. Advances in Parasitology, 2016,93:397-428. DOI:10.1016/bs.apar.2016.02.012 doi: 10.1016/bs.apar.2016.02.012

[8]	BOBARDT S D, DILLMAN A R, NAIR M G. The two faces of nematode infection: virulence and immuno-modulatory molecules from nematode parasites of mammals, insects and plants. Frontiers in Microbiology, 2020,11:577846. DOI:10.3389/fmicb.2020.577846 doi: 10.3389/fmicb.2020.577846

[9]	EHSAN M, HU R S, LIANG Q L, et al. Advances in the development of anti-Haemonchus contortus vaccines: challenges, opportunities, and perspectives. Vaccines, 2020,8(3):555. DOI:10.3390/vaccines8030555 doi: 10.3390/vaccines8030555

[10]	LAING R, KIKUCHI T, MARTINELLI A, et al. The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery. Genome Biology, 2013,14(8):R88. DOI:10.1186/gb-2013-14-8-r88 doi: 10.1186/gb-2013-14-8-r88

[11]	SCHWARZ E M, KORHONEN P K, CAMPBELL B E, et al. The genome and developmental transcriptome of the strongylid nematode Haemonchus contortus. Genome Biology, 2013,14(8):R89. DOI:10.1186/gb-2013-14-8-r89 doi: 10.1186/gb-2013-14-8-r89

[12]	PFLEIDERER G VON, ZWILLING R, SONNEBORN H H. Zur evolution der endopeptidasen, Ⅲ. Eine protease vom Molekulargewicht 11000 und eine trypsin?hnliche Fraktion aus Astacus fluviatilis Fabr. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie, 1967,348(10):1319-1331. DOI:10.1515/bchm2.1967.348.1.1319 doi: 10.1515/bchm2.1967.348.1.1319

[13]	REDDI A H. BMP-1: resurrection as procollagen C-proteinase. Science, 1996,271(5248):463. DOI:10.1126/science.271.5248.463 doi: 10.1126/science.271.5248.463

[14]	VADON-LE GOFF S, HULMES D J S, MOALI C. BMP-1/tolloid-like proteinases synchronize matrix assembly with growth factor activation to promote morphogenesis and tissue remodeling. Matrix Biology, 2015,44/45/46:14-23. DOI:10.1016/j.matbio.2015.02.006 doi: 10

[15]	MADAAN U, FAURE L, CHOWDHURY A, et al. Feedback regulation of BMP signaling by Caenorhabditis elegans cuticle collagens. Molecular Biology of the Cell, 2020,31(8):825-832. DOI:10.1091/mbc.E19-07-0390 doi: 10.1091/mbc.E19-07-0390

[16]	MULLINS M C. Holy tolloido: tolloid cleaves SOG/chordin to free DPP/BMPs. Trends in Genetics, 1998,14(4):127-129. DOI:10.1016/S0168-9525(98)01431-0 doi: 10.1016/S0168-9525(98)01431-0

[17]	GE G X, HOPKINS D R, HO W B. GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells. Molecular and Cellular Biology, 2005,25(14):5846-5858. DOI:10.1128/MCB.25.14.5846-5858.2005 doi: 10.1128/MCB.25.14.5846-5858.2005

[18]	M?EHRLEN F, HUTTER H, ZWILLING R. The astacin protein family in Caenorhabditis elegans. European Journal of Biochemistry, 2003,270(24):4909-4920. DOI:10.1046/j.1432-1033.2003.03891.x doi: 10.1046/j.1432-1033.2003.03891.x

[19]	HISHIDA R, ISHIHARA T, KONDO K, et al. hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1. The EMBO Journal, 1996,15(16):4111-4122. DOI:10.1002/j.1460-2075.1996.tb00786.x doi: 10.1002/j.1460-2075.1996.tb00786.x

[20]	STEPEK G, MCCORMACK G, PAGE A P. Collagen processing and cuticle formation is catalysed by the astacin metalloprotease DPY-31 in free-living and parasitic nematodes. International Journal for Parasitology, 2010,40(5):533-542. DOI:10.1016/j.ijpara.2009.10.007 doi: 10.1016/j.ijpara.2009.10.007

[21]	DAVIS M W, BIRNIE A J, CHAN A C, et al. A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans. Development, 2004,131(23):6001-6008. DOI:10.1242/dev.01454 doi: 10.1242/dev.01454

[22]	YAN B L, GUO X L, ZHOU Q J, et al. Hc-fau, a novel gene regulating diapause in the nematode parasite Haemonchus contortus. International Journal for Parasitology, 2014,44(11):775-786. DOI:10.1016/j.ijpara.2014.05.011 doi: 10.1016/j.ijpara.2014.05.011

[23]	DING H J, SHI H Z, SHI Y, et al. Characterization and function analysis of a novel gene, Hc-maoc-1, in the parasitic nematode Haemonochus contortus. Parasites & Vectors, 2017,10(1):67. DOI:10.1186/s13071-017-1991-1 doi: 10.1186/s13071-017-1991-1

[24]	ZHANG L, MOU L Y, CHEN X Q, et al. 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. DOI:10.1186/s13071-018-3005-3 doi: 10.1186/s13071-018-3005-3

[25]	李国清.兽医寄生虫学.北京:中国农业大学出版社,2006:200-203. DOI:10.5152/tjg.2021.20293 LI G Q. Veterinary Parasitology. Beijing: China Agricultural University Press, 2006:200-203. (in Chinese) doi: 10.5152/tjg.2021.20293

[26]	NAEEM M, IQBAL Z, ROOHI N. Ovine haemonchosis: a review. Tropical Animal Health and Production, 2021,53(1):19. DOI:10.1007/s11250-020-02439-8 doi: 10.1007/s11250-020-02439-8

[27]	STEPEK G, MCCORMACK G, BIRNIE A J, et al. The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes. Parasitology, 2011,138(2):237-248. DOI:10.1017/S0031182010001113 doi: 10.1017/S0031182010001113

[28]	YANG Y, GUO X L, CHEN X Q, et al. Functional characterization of a novel gene, Hc-dhs-28 and its role in protecting the host after Haemonchus contortus infection through regulation of diapause formation. International Journal for Parasitology, 2020,50(12):945-957. DOI:10.1016/j.ijpara.2020.04.013 doi: 10.1016/j.ijpara.2020.04.013

[29]	MIAO R, LI M J, ZHANG Q Q, et al. An ECM-to-Nucleus signaling pathway activates lysosomes for C. elegans larval development. Developmental Cell, 2020,52(1):21-37. DOI:10.1016/j.devcel.2019.10.020 doi: 10.1016/j.devcel.2019.10.020

[30]	LA?ETI? V, FAY D S. Molting in C. elegans. Worm, 2017,6(1):e1330246. DOI:10.1080/21624054.2017.1330246 doi: 10.1080/21624054.2017.1330246

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