豆状囊尾蚴丝氨酸蛋白酶抑制剂（serpin）的多抗制备、组织分布及活性研究

doi:10.12140/j.issn.1000-7423.2020.05.011

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (5): 595-601.doi: 10.12140/j.issn.1000-7423.2020.05.011

豆状囊尾蚴丝氨酸蛋白酶抑制剂（serpin）的多抗制备、组织分布及活性研究

王莉杰(), 王立群, 刘婷丽, 张少华, 刘光学, 李艳萍, 梁盼红, 骆学农^*()

中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点开放实验室,甘肃省动物寄生虫病重点实验室,兰州 730046

收稿日期:2020-03-10 出版日期:2020-10-30 发布日期:2020-11-12
通讯作者: 骆学农
作者简介:王莉杰（1991-）,女,硕士研究生,从事寄生虫分子生物学与免疫学研究。E-mail:879324102@qq.com
基金资助:
国家自然科学基金(31772726);国家自然科学基金(31372433);国家自然科学基金(1610312020017)

Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis

WANG Li-jie(), WANG Li-qun, LIU Ting-li, ZHANG Shao-hua, LIU Guang-xue, LI Yan-ping, LIANG Pan-hong, LUO Xue-nong^*()

Chinese Academy of Agricultural Sciences, Lanzhou Veterinary Research Institute, State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Public Health, Ministry of Agriculture/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute, Lanzhou 730046, China

Received:2020-03-10 Online:2020-10-30 Published:2020-11-12
Contact: LUO Xue-nong
Supported by:
National Natural Science Foundation of China(31772726);National Natural Science Foundation of China(31372433);National Natural Science Foundation of China(1610312020017)

摘要/Abstract

摘要：

目的研究豆状囊尾蚴丝氨酸蛋白酶抑制剂（Cpserpin）的组织分布及其对丝氨酸蛋白酶的抑制效果。方法利用原核表达的重组Cpserpin（rCpserpin）免疫家兔,采用间接ELISA检测免疫后血清的效价,取免疫兔血清,制备抗体并纯化后,采用Western blotting对多克隆抗体进行特异性分析,通过免疫组化试验分别观察rCpserpin在豆状带绦虫成虫和幼虫中的组织分布。构建重组质粒pPIC9K-Cpserpin,线性化后转化至毕赤酵母KM71感受态细胞,用G418筛选的高拷贝菌株进行诱导表达并纯化rCpserpin,采用发色底物法检测不同浓度（6、9、18、21 μg/ml）rCpserpin对0.5 μg/ml α-糜蛋白酶、胰蛋白酶和弹性蛋白酶等3种丝氨酸蛋白酶活性的抑制效果。结果间接ELISA检测结果显示,免疫血清多抗IgG抗体的效价达1 ∶ 51 200。Western blotting分析结果显示,纯化后的多克隆抗体可与rCpserpin和兔豆状囊尾蚴虫体可溶性抗原产生特异性反应。免疫组化试验结果显示,Cpserpin在囊尾蚴阶段表达量很低,但在成节和孕节中高度表达,广泛分布于虫体体壁和虫卵中。酵母表达的rCpserpin随浓度增加对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶的抑制效果分别增强,在21 μg/ml浓度时,对3种丝氨酸蛋白酶的抑制率最高,分别为（50.5 ± 2.5）%、（71.5 ± 1.5）%和（77.4 ± 1.5）%（均P < 0.05）。结论 rCpserpin多克隆抗体具有特异性,在豆状囊尾蚴成节和孕节中高表达,且其对α-糜蛋白酶、胰蛋白酶和弹性蛋白酶有较强的抑制作用。

关键词: 豆状囊尾蚴, 丝氨酸蛋白酶抑制剂, 组织分布, 酶活性

Abstract:

Objective To study the tissue distribution of serine protease inhibitor of Cysticercus pisiformis (Cpserpin) and its inhibitory effect on serine proteases. Methods Prokaryotically expressed recombinant Cpserpin (rCpserpin) was used to immunize rabbits, and the titers of rabbit serum after immunization were determined by indirect ELISA. The polyclonal antibodies were purified and analyzed for the specificity by Western blotting. The tissue distributions of rCpserpin in C. pisiformis adult and larva were assayed by immunohistochemistry. The recombinant plasmid pPIC9K-Cpserpin was constructed, transformed into Pichia pastoris KM71 competent cells after linearization, induced for expression in high-copy strains obtained by G418 screening, and then the products were purified. The purified recombinant protein was tested using chromogenic substrate method for its inhibitory effects on the activity of three serine proteases including α-chymotrypsin, trypsin and elastase, at the concentrations of 6, 9, 18 and 21 μg/ml, respectively. Results The results of indirect ELISA showed that the titer of serum polyclonal antibody IgG reached 1 ∶ 51 200. Western blotting showed that the purified polyclonal antibody could specifically react with rCpserpin and the C. pisiformis worm soluble antigen. Immunohistochemistry showed that Cpserpin was expressed at a low level during the cysticercus stage, but was highly expressed in mature proglottid and gravid proglottid, with wide distribution in the worm body wall and eggs. The rCpserpin expressed by P. pastoris showed enhanced inhibitiory effect on the three n of serine proteases with increased concentration, having highest inhibition rate at 21 μg/ml for α-chymotrypsin (50.5 ± 2.5)%, trypsin (71.5 ± 1.5)% and elastase (77.4 ± 1.5)% (P < 0.05). Conclusion The polyclonal antibody produced displays specificity against rCpserpin, the Cpserpin is highly expressed in mature proglottid and gravid proglottid of C. pisiformis, and the recombinant Cpserpin exhibits strong inhibitory effect on α-chymotrypsin, trypsin, and elastase.

Key words: Cysticercus pisiformis, Serine proteinase inhibitor (serpin), Tissue localization, Enzyme activity

中图分类号:

R383.32

王莉杰, 王立群, 刘婷丽, 张少华, 刘光学, 李艳萍, 梁盼红, 骆学农. 豆状囊尾蚴丝氨酸蛋白酶抑制剂（serpin）的多抗制备、组织分布及活性研究[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 595-601.

WANG Li-jie, WANG Li-qun, LIU Ting-li, ZHANG Shao-hua, LIU Guang-xue, LI Yan-ping, LIANG Pan-hong, LUO Xue-nong. Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(5): 595-601.

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参考文献 32

[1]	Zhou YX, Du AF, Zhang X J, et al. Research progress on cysticercosis pisiformis in China[J]. Chin J Ra F, 2006(1):25-28. (in Chinese)
	( 周永学, 杜爱芳, 张雪娟, 等. 我国兔豆状囊尾蚴病的研究进展[J]. 中国养兔, 2006(1):25-28.)
[2]	Zhang JH, Jiang HQ, Jiang SW. Prevention and treatment of cysticercosis pisiformis[J]. Mod J Ani Husban and Vet Med, 2009(9):38-39. (in Chinese)
	( 张金合, 姜海清, 姜诗旺. 家兔豆状囊尾蚴病的防治[J]. 现代畜牧兽医, 2009(9):38-39.)
[3]	Yu M, Yin YZ, Guo CH, et al. Prevention and treatment of common parasitic diseases in rabbits[J]. Chin Ani Husban Vet Med, 2011,38(5):190-192. (in Chinese)
	( 余淼, 尹永志, 郭春华, 等. 肉兔常见寄生虫病的防治[J]. 中国畜牧兽医, 2011,38(5):190-192.)
[4]	Zhang L, Zheng GQ, Zheng J, et al. Epidemic and prevention of commercial rabbit cysticercosis pisiformis[J]. Chin J Vet Parasitol, 2007,15(5):49-50. (in Chinese)
	( 张玲, 郑国清, 郑娟. 商品兔豆状囊尾蚴病的流行及防治[J]. 中国兽医寄生虫, 2007,15(5):49-50.)
[5]	Xu W. Diagnosis and treatment of cysticercosis pisiformis[J]. J Ani Sci and Vet Med, 2009,28(2):134. (in Chinese)
	( 许伟. 家兔囊虫病的诊断与防治[J]. 畜牧兽医杂志, 2009,28(4):134.)
[6]	Zhang SH, Luo XN, Li XQ, et al. Cloning of Taenia pisiformis actin gene and assessment of its use as an internal control[J]. Chin J Parasitol Parasit Dis, 2016,34(1):32-37. (in Chinese)
	( 张少华, 骆学农, 李雪强, 等. 豆状带绦虫肌动蛋白基因克隆及其作为分子内参的应用[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(1):32-37.)
[7]	Van Gent, Sharp P, Morgan K, et al. Serpins: structure, function and molecular evolution[J]. Int J Biochem Cell Biol, 2003,35(11):1536-1547. doi: 10.1016/s1357-2725(03)00134-1 pmid: 12824063
[8]	Irving JA, Pike RN, Lesk AM, et al. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function[J]. Genome Res, 2000,10(12):1845-1864. doi: 10.1101/gr.gr-1478r pmid: 11116082
[9]	O’Donnell RA, Blackman MJ. The role of malaria merozoite proteases in red blood cell invasion[J]. Curr Opin Microbiol, 2005,8(4):422-427. doi: 10.1016/j.mib.2005.06.018 pmid: 16019257
[10]	Debrock S, Declerck PJ. Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms[J]. Biochem Biophy Acta, 1997,1337(2):257266.
[11]	Sugino M, Imamura S, Mulenga A, et al. A serine proteinase inhibitor(serpin) from ixodid tick Haemaphysalis longicornis: cloning and preliminary assessment of its suitability as a candidate for a tick vaccine[J]. Vaccine, 2003,21(21/22):2844-2851.
[12]	Congote LF. Serpin A1 and CD91 as host instruments against HIV-1 infection: are extracellular antiviral peptides acting as intracellular messengers?[J]. Virus Res, 2007,125(2):119134.
[13]	Thorgersen EB, Ghebremariam YT, Thurman JM, et al. Candidate inhibitors of porcine complement[J]. Mol Immunol, 2007,44(8):1827-1834. doi: 10.1016/j.molimm.2006.10.004 pmid: 17109963
[14]	Hawley JH, Peanasky RJ. Ascaris suum: are trypsin inhibitors involved in species specificity of Ascarid nematodes?[J]. Exp Parasitol, 1992,75(1):112-118. doi: 10.1016/0014-4894(92)90126-u pmid: 1639157
[15]	Milstone AM, Harrison LM, Bungiro RD, et al. A broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum[J]. J Biol Chem, 2000,275(38):29391-2939. doi: 10.1074/jbc.M002715200 pmid: 10893410
[16]	Song YY, Zhang Y, Ren HN, et al. Characterization of a serine protease inhibitor from Trichinella spiralis and its participation in larval invasion of host’s intestinal epithelial cells[J]. Parasit Vector, 2018,11:499.
[17]	Merckelbach A, Ruppel A. Biochemical properties of an intracellular serpin from Echinococcus multilocularis[J]. Mol Biochem Parasitol, 2007,156(1):84-88. doi: 10.1016/j.molbiopara.2007.07.013 pmid: 17727977
[18]	Liu GX, Zhang SH, Guo AJ, et al. Identification, expression and antigenicity analysis of serpin B6 of Taenia solium[J]. Chin J Parasitol Parasit Dis, 2016,34(4):334-338, 345. (in Chinese)
	( 刘光学, 张少华, 郭爱疆, 等. 猪带绦虫丝氨酸蛋白酶抑制剂B6基因的鉴定、表达及抗原性分析[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(4):334-338, 345.)
[19]	Wang LJ, Zhang SH, Mao L, et al. Development of indirect ELISA for the diagnosis of Cysticercus pisiformis infection based on recombinant serpin[J]. Chin Vet Sci, 2018,48(4):443-449. (in Chinese)
	( 王莉杰, 张少华, 毛立, 等. 基于serpin重组蛋白的豆状囊尾蚴病间接ELISA诊断方法的初步建立[J]. 中国兽医科学, 2018,48(4):443-449.)
[20]	Li H, Peng LF. Research advances on the inhibitors of serine protease in parasitic nematodes[J]. Int J Med Parasit Dis, 2011,39(6):344-349. (in Chinese)
	李晖, 彭礼飞. 寄生线虫丝氨酸蛋白酶抑制剂研究进展[J]. 国际医学寄生虫病杂志, 2011,39(6):344-349.
[21]	Gettins PG. Serpin structure, mechanism, and function[J]. Chem Rev, 2002,102(12):4751-4804. doi: 10.1021/cr010170+ pmid: 12475206
[22]	Huang W, Haas TA, Biesterfeldt J, et al. Purification and crystallization of a novel membrane-anchored protein: the Schistosoma haematobium serpin[J]. Acta Crystrllogr D Biol Crystrllogr, 1999,55(Pt 1):350-352.
[23]	Yan Y, Liu S, Song G, et al. Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum (Sjserpin)[J]. Vet Parasitol, 2005,131(1/2):53-60.
[24]	Chopin V, Matias I, Stefano GB, et al. Amino acid sequence determination and biological activity of therin, a naturally occuring specific trypsin inhibitor from the leech Theromyzon tessulatum[J]. Eur J Biochem, 1998,254(3):565-570. doi: 10.1046/j.1432-1327.1998.2540565.x pmid: 9688267
[25]	Huang K, Strynadka NC, Bernard VD, et al. The molecular structure of the complex of Ascaris chymotrypsin/elastase inhibitor with porcine elastase[J]. Structure, 1994,2(7):679-689. doi: 10.1016/s0969-2126(00)00068-x pmid: 7922044
[26]	Ghendler Y, Amon R, Fishelson Z, et al. Schistosoma mansoni: isolation and characterization of Smpi 56, a novel serine protease inhibitor[J]. Exp Parasitol, 1994,78(2):121-131. doi: 10.1006/expr.1994.1013 pmid: 8119369
[27]	Yan Y, Hu D, Wang L, et al. Comparison of two serpins of Clonorchis sinensis by bioinformatics, expression, and localization in metacercaria[J]. Pathog Glob Health, 2014,108(4):179-185. pmid: 24831344
[28]	Hwang JH, Lee WG, Na BK, et al. Identification and characterization of a serine protease inhibitor of Paragonimus westermani[J]. Parasitol Res, 2009,104(3):244-253.
[29]	Situ YL, Shao Z, Deng L, et al. Study on the protease inhibitory activity of TtSerpin1, a serine protease inhibitor from Trichuris trichiura[J]. Chin J Parasitol Parasit Dis, 2017,35(4):342-346. (in Chinese)
	( 司徒永立, 邵正, 邓莉, 等. 毛首鞭形线虫丝氨酸蛋白酶抑制剂TtSerpin1对蛋白酶的抑制作用[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(4):342-346.)
[30]	Damasceno LM, Huang CJ, Batt CA, et al. Protein secretion in Pichia pastoris and advances in protein production[J]. Appl Microbiol Blot, 2012,93(1):31-39.
[31]	Staley CA, Huang A, Nattestad M, et al. Analysis of the 5′untranslated region (5′UTR) of the alcohol oxdiase1(AOX1) gene in recombinant protein expression in Pichia pastoris[J]. Gene, 2012,496(2):118-127. doi: 10.1016/j.gene.2012.01.006 pmid: 22285974
[32]	Kim SJ, Lee JA, Kim YH, et al. Optimization of the functional expression of Coprinus cinereus peroxidase in Pichia pastoris by varying the host and promoter[J]. J Microbiol Biotechn, 2009,19(9):966-971.

豆状囊尾蚴丝氨酸蛋白酶抑制剂（serpin）的多抗制备、组织分布及活性研究

Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis

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