捻转血矛线虫脂肪酶基因表达与生物学特性分析

doi:10.11843/j.issn.0366-6964.2018.03.015

摘要/Abstract

摘要：

选取捻转血矛线虫脂肪酶（Lipase）进行基因克隆、表达并对其生物学特性进行探究。PCR克隆捻转血矛线虫lipase基因，构建表达载体，获得融合蛋白质，采用Western blot检测该蛋白质的抗原特性；酯类酶解反应检测该融合蛋白质脂肪酶活性；实时定量PCR检测该基因在捻转血矛线虫不同发育阶段的转录情况；间接免疫荧光试验检验该抗原蛋白质在虫体组织的定位情况。结果表明，去除lipase基因信号肽序列后，基因片段长864 bp，融合蛋白质大小50 ku；该融合蛋白质在37℃左右、pH为7的条件下酶活力最高；雄性成虫lipase转录量相对虫体其他阶段最高；该抗原蛋白质主要表达于虫体表皮和肠腔。Lipase具有较好的抗原性，可能与捻转血矛线虫发育和宿主免疫调节有关，深入研究捻转血矛线虫Lipase的特性对寻找新的免疫保护性抗原和药物靶标具有意义。

Abstract:

To study the biological character of Lipase from Haemonchus contortus, the lipase gene of H. contortus preserved in our laboratory was cloned by PCR, and then expressed in procaryotic expression system. The expression vector was constructed to obtain the fusion protein. The antigenicity of the protein was analyzed by Western blot. The Lipase activity of the fusion protein was detected by esterase reaction. The location of the protein was tested by the indirect immunofluorescence. The transcription of lipase in different stages of H. contortus was detected by real-time quantification PCR. The results showed that the gene fragment encoding Lipase (without signal peptide region) sized as 864 bp. The recombinant protein weighted as 50 kD was fusion expressed. The fusion protein had highest lipase enzyme activity under 37℃ and pH7. Hc-Lipase was localized at outer and inter membrane as well as the intestinal tracts of adult worms.The expression of Hc-Lipase was higher in the male than other stages. It is indicated that Lipase from H. contortus could play important roles in worm development and modulation of the host immunity. Therefore, it is expected that Hc-Lipase could be a new target in prevention and therapy of Haemonchosis.

中图分类号:

S852.731

周丽娜, 胡孟娟, 孙伟, 李祥瑞, 徐立新, 宋小凯, 严若峰. 捻转血矛线虫脂肪酶基因表达与生物学特性分析[J]. 畜牧兽医学报, 2018, 49(3): 580-587.

ZHOU Li-na, HU Meng-juan, SUN Wei, LI Xiang-rui, XU Li-xin, SONG Xiao-kai, YAN Ruo-feng. Gene Cloning and Biological Characteristics of Lipase Gene from Haemonchus contortus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(3): 580-587.

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参考文献

[1] EMERY D L, HUNT P W, LE JAMBRE L F. Haemonchus contortus:the then and now, and where to from here?[J]. Int J Parasitol, 2016, 46(12):755-769.
[2] SIMPSON H V. Pathophysiology of abomasal parasitism:is the host or parasite responsible?[J]. Vet J, 2000, 160(3):177-191.
[3] 张家峥, 刘晓燕, 张华. 羊的捻转血矛线虫病的危害及防治[J]. 兽药市场指南, 2005(7):34-35.
ZHANG J Z, LIU X Y, ZHANG H. The damage and control of Haemonchus contortus in goat[J]. The Marking Guideline of Veterinary Drug, 2005(7):34-35. (in Chinese)
[4] YE J L, ZHU A D, TAO N G, et al. Comprehensive analysis of expressed sequence tags from the pulp of the red mutant ‘Cara Cara’ navel orange (Citrus sinensis Osbeck)[J]. J Integr Plant Biol, 2010, 52(10):856-867.
[5] KAMINSKY R, DUCRAY P, JUNG M, et al. A new class of anthelmintics effective against drug-resistant nematodes[J]. Nature, 2008, 452(7184):176-180.
[6] 孙伟. 捻转血矛线虫HC29、Dim-1和Lipase基因的克隆及免疫保护作用研究[D]. 南京:南京农业大学, 2011.
SUN W. Cloning and immunolprotection of HC29, Dim-1 and Lipase from Haemonchus contortus[D]. Nanjing:Nanjing Agricultural University, 2011. (in Chinese)
[7] ALBA-HURTADO F, MUÑOZ-GUZMÁN M A. Immune responses associated with resistance to Haemonchosis in sheep[J]. BioMed Res Int, 2013, 2013:162158.
[8] JASMER D P, ROTH J, MYLER P J. Cathepsin B-like cysteine proteases and Caenorhabditis elegans homologues dominate gene products expressed in adult Haemonchus contortus intestine[J]. Mol Biochem Parasitol, 2001, 116(2):159-169.
[9] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the method[J]. Methods, 2001, 25(4):402-408.
[10] CHEN J. Parasitoligy[M]. London:Taylor & Francis, 2000.
[11] 邹继新, 刘云. 绵羊消化道线虫病的防治措施[J]. 中国畜牧兽医文摘, 2013, 29(10):90.
ZOU J X, LIU Y. Prevention and control measures of intestinal helminthasis in sheep[J]. Chinese Digest of Animal and Veterinary Science, 2013, 29(10):90. (in Chinese)
[12] HARNETT W. Secretory products of helminth parasites as immunomodulators[J]. Mol Biochem Parasitol, 2014, 195(2):130-136.
[13] GADAHI J A, LI B J, EHSAN M, et al. Recombinant Haemonchus contortus 24 kDa excretory/secretory protein (rHcES-24) modulate the immune functions of goat PBMCs in vitro[J]. Oncotarget, 2016, 7(51):83926-83937.
[14] SATOMURA A, KURODA K, UEDA M. Generation of a functionally distinct Rhizopus oryzae lipase through protein folding memory[J]. PLoS One, 2015, 10(5):e0124545, doi:10.1371/journal.pone.0124545.
[15] BISWAS L, BISWAS R, NERZ C, et al. Role of the twin-arginine translocation pathway in Staphylococcus[J]. J Bacteriol, 2009, 191(19):5921-5929, doi:10.1128/JB.00642-09.
[16] 孟刚. 磁性纳米颗粒的制备和表征及在脂肪酶固定化方面的研究[D]. 北京:中国石油大学, 2009.
MENG G. Preparation and characterization of magnetic nanoparticles and its application in the immobilization of lipase[D]. Beijing:China University of Petroleum, 2009. (in Chinese)
[17] 白燕, 王维新. 刺参肠道蛋白酶、淀粉酶、脂肪酶与纤维素酶活性的测定方法[J]. 饲料工业, 2012, 33(20):28-32.
BAI Y, WANG W X. The methods of the activity about protease, amylase, lipase and cellilase[J]. Feed Industry, 2012, 33(20):28-32. (in Chinese)
[18] 杨汉春. 动物免疫学[M]. 2版. 北京:中国农业大学出版社, 2003:10.
YANG H C. Animal immunology[M]. 2nd ed. Beijing:China Agricultural University Press, 2003:10. (in Chinese)
[19] BUSTIN S A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):trends and problems[J]. J Mol Endocrinol, 2002, 29(1):23-39.
[20] SCHNIEDER T, HEISE M, EPE C. Genus-specific PCR for the differentiation of eggs or larvae from gastrointestinal nematodes of ruminants[J]. Parasitol Res, 1999, 85(11):895-898.
[21] 张志凯. 捻转血矛线虫新基因Hcher-1和HcMSP的克隆与阶段性表达特性分析[D]. 南京:南京农业大学, 2011.
ZHANG Z K. Cloning and differential expression analysis of Hcher-1 and HcMSP genes in Haemonchus contortus[D]. Nanjing:Nanjing Agricultural University, 2011. (in Chinese)
[22] 王小环, 杨莲如, 赵林立, 等. 免疫荧光检测技术及其在寄生虫检测中的应用进展[J]. 中国畜牧兽医, 2012, 39(3):81-84.
WANG X H, YANG L R, ZHAO L L, et al. Brief introduction of immunefluorescent test technology and application progress on parasites detection[J] China Animal Husbandry & Veterinary Medicine, 2012, 39(3):81-84. (in Chinese)
[23] NAGARAJ S H, HARSHA H C, REVERTER A, et al. Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep[J]. J Proteomics, 2012, 75(7):2141-2152.
[24] 马春晓. 捻转血矛线虫精氨酸激酶的克隆表达及其抗原特性分析[D]. 南京:南京农业大学, 2014.
MA C X. Arginine kinase in Haemonchus contortus:cloning, expression and antigenic characteristics analysis[D]. Nanjing:Nanjing Agricultural University, 2014. (in Chinese)
[25] 牛延萍. 捻转血矛线虫过氧化物酶基因的克隆和5种蛋白的抗原特性分析[D]. 南京:南京农业大学, 2012.
NIU Y P. Cloning of peroxidase and antigenic characteristics analysis of five proteins in Haemonchus contortus[D]. Nanjing:Nanjing Agricultural University, 2012. (in Chinese)