犬恶丝虫天冬氨酸转氨酶基因的原核表达及分析

Prokaryotic expression and analysis of aspartate aminotransferase gene from Dirofilaria immitis

目的了解犬恶丝虫天冬氨酸转氨酶(AspAT)基因特征及其在虫体的定位,对犬恶丝虫AspAT(DiAspAT)基因进行了原核表达和免疫荧光定位分析。方法从犬恶丝虫成虫转录组数据库中筛选到DiAspAT基因序列,通过RT-PCR方法克隆DiAspAT基因,利用相关软件进行生物信息学分析;构建原核表达载体pET32a(+)-AspAT并进行诱导表达;表达产物经Ni-NTA层析柱纯化后进行Western blotting分析;通过免疫组化技术观察DiAspAT蛋白在犬恶丝虫虫体内的定位分布。结果 DiAspAT基因的ORF框长度为1 221bp,编码406个氨基酸,理论相对分子量45.731 8kDa,等电点为8.05,表达的重组蛋白约为64kDa;Western blotting显示AspAT重组蛋白能被犬恶丝虫阳性血清识别;间接免疫荧光染色结果显示,DiAspAT在犬恶丝虫的侧索、皮下组织、肌细胞、假体腔壁、子宫及肠上皮、发育的胚胎中分布。结论成功克隆和表达了DiAspAT基因,并完成DiAspAT在雌性犬恶丝虫成虫上的定位,为进一步了解犬恶丝虫的生理代谢及犬恶丝虫病的诊断和防控奠定基础。

To understand the characteristic of aspartate aminotransferase（AspAT）gene from Dirofilaria immitis（DiAspAT）and location in the worm,the gene of DiAspAT was cloned and expressed in prokaryocyte and analysed by immunofluorescence localization.DiAspAT gene sequence was selected from transcriptome of adult D.immitis and was cloned by RTPCR method and analysed by bioinformatic approaches.A prokaryotic expression vetor of pET32a（＋）-AspAT was reconstructed and then was induced expression.The expression product was purified by Ni-NTA affinity chromatography and analysed by Western blotting.Indirect immunofluorescence assay was performed to survey the distribution of DiAspAT protein in adult female D.immitis.The open reading frame of DiAspAT gene was 1 221 bp and encodes 406 amino acids,and the molecular weight of DiAspAT was 45.731 8kDa and pI=8.05.The recombinant protein was about 64 kDa.Western blotting demonstrated that the AspAT recombinant protein（rAspAT）could be recognized by dog of DiAspATdirofilariasis positive serum.Indirect immunofluorescence localization studies illustrated the DiAspAT protein localized in the lateral chord,hypodermis,muscle,pseudocoelom wall,epithelia of uterus and intestine,developing embryos.This study first successfuly clonied and expressied the DiAspAT gene,and completed locating of DiAspAT protein in adult female D.immitis.The results lay the foundation for further understanding of metabolism of D.immitis and study the diagnosis and prevention and control of dirofilariasis.