毒害艾美耳球虫氧化还原酶EnOXIO1的原核表达与定位分析

doi:10.11843/j.issn.0366-6964.2022.05.023

Abstract

Abstract: The purpose of this study was to analyze the function of EnOXIO1, an oxidoreductase came from Eimeria necatrix. Total RNA was extracted from the gametophyte of E. necatrix Yangzhou strain to amplify EnOXIO1 gene by using RT-PCR. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into E. coli BL21 (DE3). The recombinant protein was expressed by inducing with IPTG, and its antigenicity and immunofluorescence localization was analyzed. Here we show that the full length of EnOXIO1 gene was 2 535 bp. The recombinant protein had a molecular weight of about 100 ku and mainly expressed in inclusion body. The expression level was about 0.5 mg·mL^-1 bacteria. Western blot analysis showed that the recombinant protein could be specifically recognized by 6×HIS labeled monoclonal antibody, the mouse anti-recombinant proteins polyclonal antibody, the recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts, respectively. These results indicated that the recombinant protein had good antigenicity and cross-reactivity. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein mainly existed in the wall forming body in the gametocyte and participated in the formation of the oocyst wall. In this study, EnOXIO1 protein was successfully cloned and expressed, and was located on gametocytes and oocyst wall, which laid a foundation for further analysis of the molecular mechanism of EnOXIO1 protein involved in the formation of oocyst wall, and provided important target antigens for the development of a novel immune-blocking anti-coccidiosis subunit vaccine.

Key words: Eimeria necatrix, EnOXIO1, cloning and expression, immunofluorescence localization, functional identification

CLC Number:

YE Zhuang, WANG Lele, WANG Feiyan, LIU Yue, PENG Yuemei, SU Shijie, HOU Zhaofeng, XU Jinjun, TAO Jianping, LIU Dandan. Procaryotic Expression and Immunolocalization Analysis of the EnOXIO1 of Eimeria necatrix[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1553-1561.

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Procaryotic Expression and Immunolocalization Analysis of the EnOXIO1 of Eimeria necatrix

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