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

Abstract

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

CLC Number:

S852.723

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.

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

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