香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究

doi:10.11978/2021029

Abstract

Abstract:

The C-type lectin superfamily is a class of immune proteins that can recognize and bind to polysaccharides. In this study, a new type C-type lectin gene of Crassostrea hongkongensis was cloned for the first time, and the full-length sequence of the gene was obtained. The results show that the full length of ChPerlucin gene cDNA is 577 bp, including 5′-untranslated region (UTR) in 21 bp, 3′-UTR in 73 bp, and an open reading frame (ORF) in 483 bp open reading that code 160 amino-acid polypeptide. The predicted molecular mass is 18kD, and the isoelectric point is 5.95. Amino acid sequence alignment showed that the Perlucin gene of Crassostrea hongkongensis contains a conserved carbohydrate recognition domain (CLECT). Neighbor-Joining (NJ) evolutionary tree analysis showed that ChPerlucin clustered with Perlucin from other shellfish, indicating that this gene is a new member of the Perlucin family of mollusks. The results of qRT-PCR showed that ChPerlucin was broadly expressed in various tissues and during different stages of the oyster’s embryonic and larval development; the expression of ChPerlucin increased significantly after bacteria stimulation; the recombinant protein ChPerlucin was successfully expressed using Pichia pastoris eukaryotic; and we find that ChPerlucin recombinant protein has antibacterial effect. The above results indicate that ChPerlucin plays an important role in the innate immunity of Crassostrea hongkongensis.

Key words: Crassostrea hongkongensis, innate immunity, C-type lectin, ChPerlucin

CLC Number:

S944.4

ZHAO Zehui, ZHANG Aijiao, YANG Yucheng, MAO Fan, XIAO Shu, LI Jun, ZHANG Yang, XIANG Zhiming, YU Ziniu. Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis[J].Journal of Tropical Oceanography, 2022, 41(1): 42-51.

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References 28

[1]	方紫妍, 2016. 中华绒螯蟹C型凝集素(EsLecB)的抗菌功能研究[D]. 上海: 华东师范大学.
	FANG ZIYAN, 2016. The study of a C-type lectin (EsLecB) with antibacterial activity in Eriocheir sinensis[D]. Shanghai: East China Normal University. (in Chinese with English abstract)
[2]	顾圆圆, 夏正坤, 2008. 甘露糖结合凝集素的研究进展[J]. 医学研究生学报, 21(5): 537-541.
	GU YUANYUAN, XIA ZHENGKUN, 2008. Mannose-binding lectin: from bench to personalized medicine[J]. Journal of Medical Postgraduates, 21(5): 537-541. (in Chinese with English abstract)
[3]	刘逸尘, 刘丽静, 张亦陈, 等, 2012. 中国明对虾C-型凝集素基因(Fclectin)的重组表达及活性分析[J]. 水产学报, 36(10): 1493-1502. doi: 10.3724/SP.J.1231.2012.28102
	LIU YICHEN, LIU LIJING, ZHANG YICHEN, et al, 2012. Recombinant expression and functional characterization of a C-type lectin (Fclectin) from the Chinese shrimp (Fenneropenaeus chinensis)[J]. Journal of Fisheries of China, 36(10): 1493-1502. (in Chinese with English abstract) doi: 10.3724/SP.J.1231.2012.28102
[4]	农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会, 2019. 中国渔业统计年鉴2019[M]. 北京: 中国农业出版社: 139.
	Fishery Administration of the Ministry of Agriculture and Rural Areas, National Fisheries Technology Extension Center, China Society of Fisheries, 2019. China fishery statisical yearbook[M]. Beijing: China Agriculture Press: 139. (in Chinese)
[5]	孙盛明, 傅洪拓, 宣富君, 等, 2019. 日本沼虾C型凝集素结构域家族3的cDNA克隆、原核表达和定位分析[J]. 水产学报, 43(11): 2317-2326.
	SUN SHENGMING, FU HONGTUO, XUAN FUJUN, et al, 2019. Molecular cloning, prokaryotic expression and localization analysis of C-type lectin 3 (MnLec3) cDNA from Macrobrachium nipponense[J]. Journal of Fisheries of China, 43(11): 2317-2326. (in Chinese with English abstract)
[6]	王昊, 2006. 栉孔扇贝凝集素家族基因的研究[D]. 青岛: 中国科学院大学(中国科学院海洋研究所).
	WANG HAO, 2006. Research on lectin family genes of Azumapecten farreri[D]. Qingdao: University of Chinese Academy of Sciences (Institute of Oceanology, (in Chinese with English abstract)
[7]	BI JINGXIU, NING MINGXIAO, XIE XIAOJUN, et al, 2020. A typical C-type lectin, perlucin-like protein, is involved in the innate immune defense of whiteleg shrimp Litopenaeus vannamei[J]. Fish & Shellfish Immunology, 103: 293-301.
[8]	DRICKAMER K, 1992. Engineering galactose-binding activity into a C-type mannose-binding protein[J]. Nature, 360(6400): 183-186. doi: 10.1038/360183a0
[9]	DRICKAMER K, 1993. Ca²⁺-dependent carbohydrate-recognition domains in animal proteins[J]. Current Opinion in Structural Biology, 3(3): 393-400. doi: 10.1016/S0959-440X(05)80112-5
[10]	ENDO Y, NONAKA M, SAIGA H, et al, 2003. Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus[J]. The Journal of Immunology, 170(9): 4701-4707. doi: 10.4049/jimmunol.170.9.4701
[11]	HUANG MENGMENG, SONG XIAOYAN, ZHAO JIANMIN, et al, 2013. A C-type lectin (AiCTL-3) from bay scallop Argopecten irradians with mannose/galactose binding ability to bind various bacteria[J]. Gene, 531(1): 31-38. doi: 10.1016/j.gene.2013.08.042
[12]	KANIA P W, SORENSEN R R, KOCH C, et al, 2010. Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss)[J]. Fish & Shellfish Immunology, 29(6): 910-920.
[13]	LUO TIAN, YANG HAIJIE, LI FANG, et al, 2006. Purification, characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon[J]. Developmental & Comparative Immunology, 30(7): 607-617.
[14]	MANN K, WEISS I M, ANDRÉ S, et al, 2000. The amino-acid sequence of the abalone (Haliotis laevigata) nacre protein perlucin[J]. European Journal of Biochemistry, 267(16): 5257-5264. doi: 10.1046/j.1432-1327.2000.01602.x
[15]	NAKAO M, KAJIYA T, SATO Y, et al, 2006. Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)[J]. The Journal of Immunology, 177(8): 5471-5479. doi: 10.4049/jimmunol.177.8.5471
[16]	NETH O, JACK D L, DODDS A W, et al, 2000. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition[J]. Infection and Immunity, 68(2): 688-693. doi: 10.1128/IAI.68.2.688-693.2000
[17]	NI Y, TIZARD I, 1996. Lectin-carbohydrate interaction in the immune system[J]. Veterinary Immunology and Immunopathology, 55(1-3): 205-223. doi: 10.1016/S0165-2427(96)05718-2
[18]	PRESANIS J S, KOJIMA M, SIM R B, 2003. Biochemistry and genetics of mannan-binding lectin (MBL)[J]. Biochemical Society Transactions, 31(4): 748-752. doi: 10.1042/bst0310748
[19]	QU FUFA, XIANG ZHIMING, WANG FUXUAN, et al, 2015. A novel molluscan Fos gene with immune defense function identified in the Hong Kong oyster, Crassostrea hongkongensis[J]. Developmental & Comparative Immunology, 51(1): 194-201.
[20]	SUN JIE, WANG LEI, WANG BAOJIE, et al, 2008. Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology, 25(3): 290-297.
[21]	TAYLOR M E, BRICKELL P M, CRAIG R K, et al, 1989. Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein[J]. The Biochemical Journal, 262(3): 763-771. doi: 10.1042/bj2620763
[22]	TIRAPÉ A, BACQUE C, BRIZARD R, et al, 2007. Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis[J]. Developmental & Comparative Immunology, 31(9): 859-873.
[23]	WANG LINGLING, YUE FENG, SONG XIAORUI, et al, 2015. Maternal immune transfer in mollusc[J]. Developmental & Comparative Immunology, 48(2): 354-359.
[24]	WEIS W I, TAYLOR M E, DRICKAMER K, 1998. The C-type lectin superfamily in the immune system[J]. Immunological Reviews, 163(1): 19-34. doi: 10.1111/imr.1998.163.issue-1
[25]	YUE FENG, ZHOU ZHI, WANG LINGLING, et al, 2013a. Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge[J]. Developmental & Comparative Immunology, 41(4): 569-577.
[26]	YUE FENG, SHI XIAOWEI, ZHOU ZHI, et al, 2013b. The expression of immune-related genes during the ontogenesis of scallop Chlamys farreri and their response to bacterial challenge[J]. Fish & Shellfish Immunology, 34(3): 855-864.
[27]	ZHANG HAO, PEATMAN E, LIU HONG, et al, 2012. Characterization of a mannose-binding lectin from channel catfish (Ictalurus punctatus)[J]. Research in Veterinary Science, 92(3): 408-413. doi: 10.1016/j.rvsc.2011.03.024
[28]	ZHAO ZHIYING, YIN ZHIXIN, XU XIAOPENG, et al, 2009. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity[J]. Journal of Virology, 83(1): 347-356. doi: 10.1128/JVI.00707-08

引物名称	序列(5'-3')	用途
ChPerlucin F₁	GCTACTGCCAGGCTTACCATTCCAA	ChPerlucin 3′RACE引物
ChPerlucin F₂	ATAGGAATAACGGACCTTACAGCAG	ChPerlucin 3′RACE引物
ChPerlucin R₁	GTTGGATGAATACACCCACTCGTTT	ChPerlucin 5′RACE引物
ChPerlucin R₂	TAAGGTCCGTTATTCCTATCCAAAA	ChPerlucin 5′RACE引物
ChPerlucin F₃	GGAAAGAATGTAAGGTAAACAATGG	ChPerlucin ORF引物
ChPerlucin R₃	TGACGCTTGGTTAATCAATTCTTTT	ChPerlucin ORF引物
ChPerlucin F₄	CTGCCAGGCTTACCATTCCAA	ChPerlucin实时荧光定量PCR
ChPerlucin R₄	GGCTTGCCCAGTTTGTCGTCT	ChPerlucin实时荧光定量PCR
ChPerlucin F₅	AAGGATCCACCATGGGTTCCGGCTGCCGCTCGGGATGGACTCAGTACAAA	pPIC3.5K-ChPerlucin载体构建
ChPerlucin R₅	TTGAATTCTCAATGATGATGATGATGATGTCCAATGATCACCGGGCCACT	pPIC3.5K-ChPerlucin载体构建
GAPDH-F	GGATTGGCGTGGTGGTAGAG	GAPDH实时荧光定量PCR
GAPDH-R	GTATGATGCCCCTTTGTTGAGTC	GAPDH实时荧光定量PCR

Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis

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