鸡毒害艾美耳球虫ApiAP2转录因子的克隆、表达与虫体内定位

doi:10.11843/j.issn.0366-6964.2022.12.023

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (12): 4379-4388.doi: 10.11843/j.issn.0366-6964.2022.12.023

鸡毒害艾美耳球虫ApiAP2转录因子的克隆、表达与虫体内定位

王礼跃^1,2, 冯茜茜^1,2, 蔡为民^1,2, 刘丹丹^1,2, 候照峰^1,2, 康喜龙^2,3, 张知之^1,2, 范雪莲^1,2, 朱玉^1,2, 许金俊^1,2, 潘志明^2,3,4, 陶建平^1,2*

1. 扬州大学兽医学院, 扬州 225009;
2. 江苏动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009;
3. 江苏省人兽共患病学重点实验室, 扬州 225009;
4. 宿迁学院, 宿迁 223800

收稿日期:2022-06-24 出版日期:2022-12-23 发布日期:2022-12-25
通讯作者: 陶建平，主要从事兽医寄生虫病及其防治技术研究，E-mail:yzjptao@126.com
作者简介:王礼跃(1997-)，男，江苏阜宁人，硕士生，主要从事鸡球虫病研究，E-mail:961452686@qq.com；冯茜茜(1998-)，女，江苏南通人，硕士生，主要从事鸡球虫病研究，E-mail:1411064524@qq.com。王礼跃和冯茜茜为同等贡献作者
基金资助:
国家自然科学基金(31972698)；江苏省重点研发计划(现代农业，BE2021354);江苏省高校优势学科建设三期工程项目

Cloning, Expression and Localization of Transcription Factor ApiAP2 of Eimeria necatrix

WANG Liyue^1,2, FENG Qianqian^1,2, CAI Weimin^1,2, LIU Dandan^1,2, HOU Zhaofeng^1,2, KANG Xilong^2,3, ZHANG Zhizhi^1,2, FAN Xuelian^1,2, ZHU Yu^1,2, XU Jinjun^1,2, PAN Zhiming^2,3,4, TAO Jianping^1,2*

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;
3. Jiangsu Key Laboratory of Zoonosis, Yangzhou 225009, China;
4. Suqian University, Suqian 223800, China

Received:2022-06-24 Online:2022-12-23 Published:2022-12-25

摘要/Abstract

摘要： 顶复门原虫的AP2结构域蛋白(ApiAP2)可能是调控虫体生长发育的转录因子。本研究旨在克隆、表达毒害艾美耳球虫ApiAP2转录因子，检测其天然蛋白及其在虫体内的亚细胞定位。以毒害艾美耳球虫第3代裂殖子的总RNA为模板，RT-PCR扩增毒害艾美耳球虫EnApiAP2基因后，连接至pMD18-T载体，测序后构建pET28a (+)-EnApiAP2表达载体，转化至表达菌BL21(DE3)，重组菌经测序鉴定后诱导表达，并纯化复性重组蛋白。用重组蛋白免疫BALB/c小鼠，制备鼠抗rEnApiAP2多克隆抗体。利用多克隆抗体，分别用Western blot和间接免疫荧光试验检测裂殖子中天然EnApiAP2蛋白及其定位。最后，应用荧光定量PCR分析EnApiAP2基因在第2、3代裂殖子中的转录水平。结果显示，该基因全长1 830 bp，编码610个氨基酸，含有一个AP2结构域，预测分子质量67.69 ku；重组蛋白大小约为74 ku，主要以包涵体形式存在，可以被6×HIS标签单抗、鸡抗毒害艾美耳球虫康复血清和鸡抗柔嫩艾美耳球虫康复血清识别；在第3代裂殖子中检测到天然EnApiAP2蛋白，分子质量约为85 ku；EnApiAP2蛋白定位在第2、3代裂殖子细胞核内；第3代裂殖子EnApiAP2转录水平显著高于第2代裂殖子(P<0.01)。成功克隆、表达了EnApiAP2基因，证实该基因表达的蛋白定位在裂殖子的细胞核内，为进一步研究EnApiAP2蛋白的转录因子功能奠定了基础。

关键词: 毒害艾美耳球虫, EnApiAP2基因, 克隆, 表达, 定位

Abstract: Apicomplexan Ap2 domain proteins (ApiAP2) may be transcription factors that control the development and sexual differentiation of parasites. This study aims to clone and express transcription factor ApiAP2 of Eimeria necatrix, and detect the native ApiAP2 protein and its localization in the parasites. The gene (EnApiAP2) was cloned from the total RNA of the third generation merozoites (MZ-3) of E. necatrix by RT-PCR, and inserted to pMD18-T vector by TA cloning. After sequencing analysis, EnApiAP2 cDNA was subcloned to pET-28a(+) vector to obtain a recombinant prokaryotic plasmid. After transformed into expression strain BL21 (DE3), the recombinant plasmid pET28a(+)-EnApiAP2 was induced to express by IPTG, which was purified and renatured. BALB/c mice were immunized with purified recombinant protein, and the polyclonal anti-EnApiAP2 antibodies were prepared, which were used to detect the native EnApiAP2 protein and its localization in the second generation merozoites (MZ-2) and MZ-3 of E. necatrix by Western blot and indirect immunofluorescence assay, respectively. Finally, the transcriptional level of EnApiAP2 in MZ-2 and MZ-3 was analyzed by qRT-PCR. The results showed that the target gene was 1 830 bp, coding 610 amino acids with a predicated molecular weight of 67.69 ku and one AP2 domain. The recombinant protein was about 74 ku and predominately expressed in inclusion body. Western blot analysis indicated that the recombinant protein could be specifically recognized by 6×HIS tag monoclonal antibodies, the convalescent serum of chicken infected with E. necatrix or E. tenella. Native EnApiAP2 protein was detected in MZ-3 and had a molecular weight of 85 ku. EnApiAP2 protein was located in the nucleus of MZ-3. The transcription level of EnApiAP2 in MZ-3 was significantly higher than that in MZ-2 (P < 0.01). In conclusion, EnApiAP2 gene was successfully cloned and expressed. EnApiAP2 protein located in the nucleus of MZ-2 and MZ-3. These results lay a foundation for further study on the transcription factor function of EnApiAP2 protein.

Key words: Eimeria necatrix, EnApiAP2 gene, cloning, expression, localization

中图分类号:

S852.723

王礼跃, 冯茜茜, 蔡为民, 刘丹丹, 候照峰, 康喜龙, 张知之, 范雪莲, 朱玉, 许金俊, 潘志明, 陶建平. 鸡毒害艾美耳球虫ApiAP2转录因子的克隆、表达与虫体内定位[J]. 畜牧兽医学报, 2022, 53(12): 4379-4388.

WANG Liyue, FENG Qianqian, CAI Weimin, LIU Dandan, HOU Zhaofeng, KANG Xilong, ZHANG Zhizhi, FAN Xuelian, ZHU Yu, XU Jinjun, PAN Zhiming, TAO Jianping. Cloning, Expression and Localization of Transcription Factor ApiAP2 of Eimeria necatrix[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4379-4388.

参考文献 22

[1]	HAO L L, LIU X Y, ZHOU X Y, et al. Transient transfection of Eimeria tenella using yellow or red fluorescent protein as a marker[J]. Mol Biochem Parasitol, 2007, 153(2):213-215.
[2]	BURRELL A, TOMLEY F M, VAUGHAN S, et al. Life cycle stages, specific organelles and invasion mechanisms of Eimeria species[J]. Parasitology, 2020, 147(3):263-278.
[3]	CHUCK G, MEELEY R B, HAKE S. The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet 1[J]. Genes Dev, 1998, 12(8):1145-1154.
[4]	BALAJI S, BABU M M, IYER L M, et al. Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains[J]. Nucleic Acids Res, 2005, 33(13):3994-4006.
[5]	BISCHOFF E, VAQUERO C. In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum[J]. BMC Genomics, 2010, 11(1):34.
[6]	ALTSCHUL S F, WOOTTON J C, ZASLAVSKY E, et al. The construction and use of log-odds substitution scores for multiple sequence alignment[J]. PLoS Comput Biol, 2010, 6(7):e1000852.
[7]	PIESZKO M, WEIR W, GOODHEAD I, et al. ApiAP2 factors as candidate regulators of stochastic commitment to merozoite production in Theileria annulata[J]. PLoS Negl Trop Dis, 2015, 9(8):e0003933.
[8]	OBERSTALLER J, PUMPALOVA Y, SCHIELER A, et al. The Cryptosporidium parvum ApiAP2 gene family:insights into the evolution of apicomplexan AP2 regulatory systems[J]. Nucleic Acids Res, 2014, 42(13):8271-8284.
[9]	DE SILVA E K, GEHRKE A R, OLSZEWSKI K, et al. Specific DNA-binding by apicomplexan AP2 transcription factors[J]. Proc Natl Acad Sci U S A, 2008, 105(24):8393-8398.
[10]	SU S J, HOU Z F, LIU D D, et al. Comparative transcriptome analysis of second and third-generation merozoites of Eimeria necatrix[J]. Parasit Vectors, 2017, 10(1):388.
[11]	刘丹丹. 毒害艾美耳球虫配子体抗原基因的克隆表达与功能研究[D]. 扬州:扬州大学, 2014.LIU D D. Cloning, expression and function research of gametocyte antigen genes of Eimeria necatrix[D]. Yangzhou: Yangzhou University, 2014. (in Chinese)
[12]	LIU D D, CAO L Q, ZHU Y L, et al. Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation[J]. Parasit Vectors, 2014, 7:27.
[13]	SALMELA L, RIVALS E. LoRDEC:accurate and efficient long read error correction[J]. Bioinformatics, 2014, 30(24):3506-3514.
[14]	LINDNER S E, DE SILVA E K, KECK J L, et al. Structural determinants of DNA binding by a P. falciparum ApiAP2 transcriptional regulator[J]. J Mol Biol, 2010, 395(3):558-567.
[15]	JENINGA M D, QUINN J E, PETTER M. ApiAP2 transcription factors in apicomplexan parasites[J]. Pathogens, 2019, 8(2):47.
[16]	MORRISON D A, BORNSTEIN S, THEBO P, et al. The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa)[J]. Int J parasitol, 2004, 34(4):501-514.
[17]	SINGH S, SANTOS J M, ORCHARD L M, et al. The PfAP2-G2 transcription factor is a critical regulator of gametocyte maturation[J]. Mol Microbiol, 2021, 115(5):1005-1024.
[18]	WALKER R, GISSOT M, CROKEN M M, et al. The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation[J]. Mol Microbiol, 2013, 87(1):641-655.
[19]	HONG D P, RADKE J B, WHITE M W. Opposing transcriptional mechanisms regulate Toxoplasma development[J]. mSphere, 2017, 2(1):e00347-16.
[20]	HUANG S, HOLMES M J, RADKE J B, et al. Toxoplasma gondii AP2IX-4 regulates gene expression during bradyzoite development[J]. mSphere, 2017, 2(2):e00054-17.
[21]	SHANG X M, SHEN S J, TANG J X, et al. A cascade of transcriptional repression determines sexual commitment and development in Plasmodium falciparum[J]. Nucleic Acids Res, 2021, 49(16):9264-9279.
[22]	CARRINGTON E, COOIJMANS R H M, KELLER D, et al. The ApiAP2 factor PfAP2-HC is an integral component of heterochromatin in the malaria parasite Plasmodium falciparum[J]. iScience, 2021, 24(5):102444.

鸡毒害艾美耳球虫ApiAP2转录因子的克隆、表达与虫体内定位

Cloning, Expression and Localization of Transcription Factor ApiAP2 of Eimeria necatrix

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