火鸡组织滴虫α-actinin1蛋白的原核表达及其在虫体中的定位分析

Prokaryotic expression and localization analysis of α-actinin1 protein of Histomonas meleagridis

为研究火鸡组织滴虫(Histomonas meleagridis)α-actinin1蛋白(Hmα-actinin1)的功能,本研究根据GenBank中火鸡组织滴虫α-actinin1基因序列(FM200068)设计引物,经RT-PCR从虫体总RNA中扩增Hmα-actinin1基因,全长为1 839 bp,克隆至p ET-28a(+)构建原核表达载体后,转化BL21(DE3),IPTG诱导表达。SDS-PAGE分析显示,rHmα-actinin1蛋白以可溶形式存在,相对分子量约为73 ku。采用His标签层析法纯化重组蛋白后,常规免疫BALB/c小鼠后采集血清,利用间接ELISA检测结果显示血清抗体效价达1.102 400。Western blot分析rHmα-actinin1蛋白的抗原性,结果显示该蛋白能同时被鸡源、火鸡源火鸡组织滴虫阳性血清及鼠抗rHmα-actinin1阳性血清特异性识别。利用间接免疫荧光检测天然蛋白Hmα-actinin1在虫体中的分布,结果显示该蛋白主要分布于虫体的胞质内。上述结果表明,该蛋白具有很好的抗原性,本研究为火鸡组织滴虫治疗药物和疫苗研制提供了新的靶标,也为进一步研究火鸡组织滴虫致病机制奠定了基础。

In order to study the function of α-actinin1 in Histomonas meleagridis(Hmα-actinin1), primers were designed based on sequence(FM200068) in Gen Bank, Hmα-actinin1 gene with a full length of 1 839 bp was amplified from total RNA by RT-PCR and then cloned into p ET-28 a(+) to construct prokaryotic expression vector. The expressiong vector was transformed into BL21(DE3) and induced by IPTG. SDS-PAGE showed that r Hmα-actinin1 protein had a molecular weight of 73 ku. The recombinant protein was purified by His label chromatography and used to immunize mouse. The titer of antibody detected by indirect ELISA was 1.102 400. The antigenicity of r Hmα-actinin1 was analyzed by western blot. Our results showed that r Hmαactinin1 could be specifically recognized by both turkey and chicken positive serum anti-H. meleagridis and mouse positive serum anti-r Hmα-actinin1. The distributions of natural protein was analyzed by indirect immunofluorescence, showing that natural Hmα-actinin1 protein mainly located in cytoplasm. Our results indicated that this recombinant protein has good antigenicity, which may provide a new target for the development of drugs and vaccines, thus laid a foundation for the further analysis of the pathogenic mechanism of Histomonas meleagridis.