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蛋白质翻译后O-糖基化修饰普遍存在于真核生物、细菌和古细菌,多肽:N-乙酰氨基半乳糖转移酶2(polypeptide:N-acetylgalactosaminyltransferase 2,ppGalNAc-T2)是O-糖基化反应的限速酶。基因组数据分析揭示艾美耳球虫具有O-糖基化反应途径。为研究柔嫩艾美耳球虫(Eimeria tenella)蛋白质翻译后糖基化修饰及其关键酶的药靶效用,对Et ppGalNAc-T2基因进行了克隆,并检测其在E.tenella不同发育阶段的表达动态。根据EuPathDB中所预测Et ppGalNAc-T2基因序列设计引物,以RT-PCR方法扩增获得Et ppGalNAc-T2的ORF,插入酶切位点后连接到原核表达载体pGEX-6p-1进行诱导表达。提取E.tenella广东株未孢子化卵囊、孢子化7h卵囊、孢子化卵囊、子孢子和第二代裂殖子5个发育阶段的总RNA,以qRT-PCR法检测Et ppGalNAc-T2转录水平的动态变化。结果表明,Et ppGalNAc-T2的全长ORF为1 983bp,编码660个氨基酸,在E.coli Transetta(DE3)可溶性表达。qRT-PCR结果显示,Et ppGalNAc-T2转录水平随发育阶段不同而有显著差异,子孢子阶段表达水平最高、而在孢子化卵囊则几近不能检出。结果为进一步研究EtppGalNAc-T2的功能提供了试验基础。
Translational O-glycosylation of proteins is ubiquitous in eukaryotes, bacteria and archaebacteria. The polypeptide: N-acetylgalactosaminyltransferase 2 (ppGalNAc-T2) is O-glycosylation The rate-limiting enzyme. Genome data analysis revealed that Eimeria has O-glycosylation pathways. In order to study the post-translational glycosylation of Eimeria tenella protein and its target enzyme, the Et ppGalNAc-T2 gene was cloned and tested at different developmental stages of E.tenella dynamic. Primers were designed according to the predicted Et ppGalNAc-T2 gene sequence in EuPathDB. The ORF of Et ppGalNAc-T2 was amplified by RT-PCR. The ORF was inserted into the restriction site and ligated into prokaryotic expression vector pGEX-6p-1. Total RNA was extracted from E.tenella Guangdong spore-free oocysts, sporulated 7h oocysts, sporulated oocysts, sporozoites and second-generation merozoites in five developmental stages. The levels of Et ppGalNAc-T2 Dynamic changes in transcriptional level. The results showed that the full-length ORF of Et ppGalNAc-T2 was 1 983 bp, encoding 660 amino acids and was soluble in E.coli Transetta (DE3). The results of qRT-PCR showed that the transcriptional level of Et ppGalNAc-T2 was significantly different with different stages of development. The highest level of sporozoite expression was detected in the sporozoite oocysts, however, almost no detection was found. The results provide experimental basis for further study on the function of EtppGalNAc-T2.