毒害艾美耳球虫钙调素依赖性蛋白激酶基因的原核表达及其在不同发育阶段的表达分析

Prokaryotic Expression of Ca2+/Calmodulin-dependent Kinase（CaMK） Gene and Expression Analysis at Different Development Stages of Eimeria necatrix

在前期分析比较毒害艾美耳球虫第二、三代裂殖子和配子体的转录组时,发现基因代号为ENH00078000的钙调素依赖性蛋白激酶(CaMK)基因呈差异表达。为了研究毒害艾美耳球虫钙调素依赖性蛋白激酶(EnCaMK)的功能,研究首先人工合成了该基因,构建pET-28a-EnCaMK表达载体,转化大肠杆菌BL21,经IPTG诱导表达,对表达产物进行SDS-PAGE分析和Western blot鉴定,并进一步分析EnCaMK在毒害艾美耳球虫第二、三代裂殖子和配子体中的表达情况。结果显示:重组蛋白大小为48 ku左右,以包涵体形式为多,能被组氨酸(His)单克隆抗体特异性识别和鸡抗毒害艾美耳球虫多克抗隆抗体血清识别;用抗重组蛋白的鼠多克隆抗体在第三代裂殖子和配子体蛋白中检测到35 ku左右的EnCaMK条带,且在第三代裂殖子中表达量较高,显示差异表达。研究结果为进一步研究EnCaMK的酶活性与功能奠定了基础。

The ENH00078000 gene encoding Ca^2+/calmodulin-dependent protein kinase(CaMK) was found to be differentially expressed among the second generation merozoites(MZ-2), the third generation merozoites(MZ-3) and gametocytes of Eimeria necatrix in our previous studies. In the present study, the gene was synthesized, and then the pET-28 a-EnCaMK expression vector was constructed and transformed into Escherichia coli BL21, in which the recombinant protein(rEnCaMK) was induced to express with IPTG. The expression products were identified by SDS-PAGE and Western blot, and the expression of EnCaMK in MZ-2 and MZ-3 and gametophytes of E. necatrix was further analyzed.The results showed that rEnCaMK was about 48 ku and mostly in the form of inclusion bodies. The rEnCaMK could be specifically recognized by histidine(His) monoclonal antibody and chicken anti-E. necatrix polyclonal antibody. A specific band of about 35 ku could be detected by the mouse anti-rEnCaMK polyclonal antibody in MZ-3 and gametocyte,and the band stained darker in MZ-3 than in gametocyte, which showed the EnCaMK was differentially expressed.The results laid the foundation for further study of the enzymatic activity and function of EnCaMK.