多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析

doi:10.11843/j.issn.0366-6964.2017.12.017

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (12): 2374-2382.doi: 10.11843/j.issn.0366-6964.2017.12.017

多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析

黄兴^1,2, 徐璟¹, 王宇¹, 杨应东³, 万洁³, 郭承¹, 古小彬¹, 谢跃¹, 杨光友^1*

1. 四川农业大学动物医学院, 成都 611130;
2. 成都农业科技职业学院, 成都 611130;
3. 攀枝花农林科学研究院, 攀枝花 617061

收稿日期:2017-06-08 出版日期:2017-12-23 发布日期:2017-12-23
通讯作者: 杨光友,教授,博士,博导,主要从事动物寄生虫病学研究,E-mail:guangyou1963@aliyun.com
作者简介:黄兴(1983-),男,土家族,重庆人,副教授,博士,主要从事动物寄生虫病学研究,E-mail:huangxing198308@163.com
基金资助:
四川省科技支撑计划项目（2015NZ1504）

Prokaryotic Expression, Tissue Localization and Enzymatic Activity Analysis of Taenia multiceps dUTPase Gene

HUANG Xing^1,2, XU Jing¹, WANG Yu¹, YANG Ying-dong³, WAN Jie³, GUO Cheng¹, GU Xiao-bin¹, XIE Yue¹, YANG Guang-you^1*

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Chengdu Agricultural College, Chengdu 611130, China;
3. Panzhihua Academy of Agricultural and Forestry Sciences, Panzhihua 617061, China

Received:2017-06-08 Online:2017-12-23 Published:2017-12-23

摘要/Abstract

摘要：

探讨多头带绦虫脱氧尿苷三磷酸酶（Tm-dUTPase）的基因特征，并初步分析Tm-dUTPase的酶学活性。通过原核表达，得到重组Tm-dUTPase蛋白，并对其进行Western blot分析、免疫荧光定位及酶学活性分析。序列分析结果显示，Tm-dUTPase基因ORF框为447 bp，编码148个氨基酸，预测的蛋白质分子大小为16.39 ku，等电点为6.263；与猪囊尾蚴和豆状囊尾蚴dUTPase基因的氨基酸序列相似性分别为97%和91%。原核表达的Tm-dUTPase为可溶性蛋白，纯化后的蛋白质质量浓度为0.907 mg·mL^-1，Western blot分析显示，该蛋白质能够被山羊脑多头蚴阳性血清所识别，具有较强的反应原性。免疫荧光定位显示Tm-dUTPase主要分布于多头带绦虫成虫的体内实质区，同时广泛分布于多头带绦虫幼虫（脑多头蚴）的头节和包囊囊壁外层。酶学活性试验测得重组Tm-dUTPase的比活力为986.29 U·mg^-1，EDTA能够抑制Tm-dUTPase活性，而金属离子Mg²⁺、Zn²⁺、Cu²⁺能够增强Tm-dUTPase活性。上述结果为进一步研究Tm-dUTPase基因的生物学功能奠定了基础。

Abstract:

The aim of this study was to characterize the dUTPase gene of Taenia multiceps and analyze its enzymatic activity. The recombinant Tm-dUTPase was expressed in prokaryotic system and the enzymatic activity was measured; Western blotting analysis and immunofluorescence localization were also performed. Sequence analysis showed that the Tm-dUTPase gene contains a 447 bp open reading frame and the mature polypeptide consists of 148 amino acid residues, with a predicted molecular weight of 16.39 kDa and PI of 6.263; the deduced amino acid of Tm-dUTPase shares 97% and 91% similarity with dUTPase from Cysticercus cellulosae and Taenia pisiformis respectively. The recombinant Tm-dUTPase was expressed as a soluble protein, and the purified protein has a concentration of 0.907 mg·mL^-1. The Western blotting showed that the recombinant Tm-dUTPase could be recognized by the serum of goat naturally infected with Coenurus cerebralis, indicating a relatively high immunoreactivity. The immunolocalization showed that the native Tm-dUTPase mainly distributed to the parenchymatous zone of the adult parasite, and widely distributed to the protoscolexes and out layer of the cystic wall of Coenurus cerebralis. The enzyme activity of Tm-dUTPase was 986.29 U·mg^-1, and the activity could be inhibited by EDTA and enhanced by Mg²⁺, Zn²⁺ and Cu²⁺. These results would greatly benefit the further study of the biological function of Tm-dUTPase.

中图分类号:

S852.734

黄兴, 徐璟, 王宇, 杨应东, 万洁, 郭承, 古小彬, 谢跃, 杨光友. 多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析[J]. 畜牧兽医学报, 2017, 48(12): 2374-2382.

HUANG Xing, XU Jing, WANG Yu, YANG Ying-dong, WAN Jie, GUO Cheng, GU Xiao-bin, XIE Yue, YANG Guang-you. Prokaryotic Expression, Tissue Localization and Enzymatic Activity Analysis of Taenia multiceps dUTPase Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(12): 2374-2382.

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参考文献

[1] EDWARDS G T, HERBERT I V. Observations on the course of Taenia multiceps infections in sheep:Clinical signs and post-mortem findings[J]. Br Vet J, 1982, 138(6):489-500.
[2] IBECHUKWU B I, ONWUKEME K E. Intraocular coenurosis:A case report[J]. Br J Ophthalmol, 1991, 75(7):430-431.
[3] CHRISTODOULOPOULOS G. Two rare clinical manifestations of coenurosis in sheep[J]. Vet Parasitol, 2007, 143(3-4):368-370.
[4] EL-ON J, SHELEF I, CAGNANO E, et al. Taenia multiceps:a rare human cestode infection in Israel[J]. Vet Ital, 2008, 44(4):621-631.
[5] GAUCI C, VURAL G, ÖNCEL T, et al. Vaccination with recombinant oncosphere antigens reduces the susceptibility of sheep to infection with Taenia multiceps[J]. Int J Parasitol, 2008, 38(8-9):1041-1050.
[6] HARRIS J M, MCINTOSH E M, MUSCAT G E O. Structure/function analysis of a dUTPase:Catalytic mechanism of a potential chemotherapeutic target[J]. J Mol Biol, 1999, 288(2):275-287.
[7] MOROZ O V, HARKIOLAKI M, GALPERIN M Y, et al. The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases[J]. J Mol Biol, 2004, 342(5):1583-1597.
[8] HARKIOLAKI M, DODSON E J, BERNIER-VILLAMOR V, et al. The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding fold[J]. Structure, 2004, 12(1):41-53.
[9] DAUTER Z, PERSSON R, ROSENGREN A M, et al. Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex[J]. J Mol Biol, 1999, 285(2):655-673.
[10] ZHAO Z, KE F, GUI J F, et al. Characterization of an early gene encoding for dUTPase in Rana grylio virus[J]. Virus Res, 2007, 123(2):128-137.
[11] HIDALGO-ZARCO F, GONZALEZ-PACNOWSKA D. Trypanosomal dUTPases as potential targets for drug design[J]. Curr Protein Pept Sci, 2001, 2(4):389-397.
[12] LADNER R D, MCNULTY D E, CARR S A, et al. Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase[J]. J Biol Chem, 1996, 271(13):7745-7751.
[13] MERCER A A, FRASER K M, STOCKWELL P A, et al. A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus[J]. Virology, 1989, 172(2):665-668.
[14] SALTARELLI M, QUERAT G, KONINGS D A M, et al. Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus[J]. Virology, 1990, 179(1):347-364.
[15] WHITTINGHAM J L, LEAL I, NGUYEN C, et al. dUTPase as a platform for antimalarial drug design:Structural basis for the selectivity of a class of nucleoside inhibitors[J]. Structure, 2005, 13(2):329-338.
[16] 刘振勇, 吕志慧, 郑亚东, 等. 猪囊尾蚴dUTPase基因的克隆、表达及其产物的酶学活性分析[J]. 中国农业科学, 2010, 43(1):192-199.
LIU Z Y, LÜ Z H, ZHENG Y D, et al. Cloning, expression of cysticercus cellulosae dUTPase and the determination of its enzymatic activity[J]. Scientia Agricultura Sinica, 2010, 43(1):192-199. (in Chinese)
[17] 陈林, 杨德英, 孙家刚, 等. 豆状带绦虫六钩蚴Tp-dUTPase基因的表达及血清学诊断效果[J]. 中国兽医学报, 2014, 34(7):1100-1105.
CHEN L, YANG D Y, SUN J G, et al. Prokaryotic expression of Tp-dUTPase gene of Taenia pisiformis oncospheres and establishment of dot-ELISA using the expressed protein[J]. Chinese Journal of Veterinary Science, 2014, 34(7):1100-1105. (in Chinese)
[18] 宋星桔, 胡丹丹, 钟秀琴, 等. 细粒棘球绦虫铜锌超氧化物歧化酶基因的表达与特征分析[J]. 畜牧兽医学报, 2016, 47(2):346-353.
SONG X J, HU D D, ZHONG X Q, et al. Prokaryotic expression and characterization of Cu/Zn superoxide dismutase of Echinococcus granulosus[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(2):346-353. (in Chinese)
[19] GRINDEY G B, NICHOL C A. Micro procedure for determination of pyrophosphate and orthophosphate[J]. Anal Biochem, 1970, 33(1):114-119.
[20] GRINDEY G B, NICHOL C A. Mammalian deoxyuridine 5'-triphosphate pyrophosphatase[J]. Biochim Biophys Acta, 1971, 240(2):180-183.
[21] GOULIAN M, BLEILE B, TSENG B Y. The effect of methotrexate on levels of dUTP in animal cells[J]. J Biol Chem, 1980, 255(22):10630-10637.
[22] MAFOJANE N A, APPLETON C C, KRECEK R C, et al. The current status of neurocysticercosis in Eastern and Southern Africa[J]. Acta Trop, 2003, 87(1):25-33.
[23] 陈巧林, 任宏伟, 康巧华, 等. 人细胞核dUTPase的克隆表达及其酶学活性[J]. 中国生物化学与分子生物学报, 2003, 19(5):606-611.
CHEN Q L, REN H W, KANG Q H, et al. Cloning, expression of human nucleolus dUTPase and the determination of its enzymatic activity[J]. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(5):606-611. (in Chinese)

多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析

Prokaryotic Expression, Tissue Localization and Enzymatic Activity Analysis of Taenia multiceps dUTPase Gene

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