鸡毒害艾美耳球虫ApiAP2转录因子的克隆、表达与虫体内定位

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。

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

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.