刀额新对虾精氨酸激酶基因的克隆与真核表达

Cloning and eukaryotic expression of arginine kinase gene of Metapenaeus ensis

目的克隆刀额新对虾精氨酸激酶的基因并进行真核表达。方法提取刀额新对虾总RNA,经逆转录获得精氨酸激酶的cDNA,插入质粒pPIC9K构建重组真核表达质粒,转化毕赤酵母进行诱导表达,用SDS-PAGE和斑点免疫分析鉴定重组表达产物。结果逆转录获得的精氨酸激酶cDNA全长1071bp,DNA序列分析结果与GenBank收录的序列(EU497674)比较,同源性为99.5%;重组表达质粒转化酵母的表达产物经SDS-PAGE分析,其相对分子质量约40kD;表达产物与虾过敏者血清呈较强反应性。结论克隆得到刀额新对虾的精氨酸激酶基因并获得其重组真核表达产物,为进一步研制虾类食物过敏症的相关检测试剂提供了试验依据。

Objective Cloning and eukaryotic expression of arginine kinase （AK） gene of Metapenaeus en- sis. Methods The total mRNA was extracted from muscle tissue of Metapenaeus ensis and the cDNA of AK gene was expanded with reverse PCR. A recombinant eukaryotic expression vector was constructed by inserting the expanded AK cDNA into a plasmid pPICgK and transferred into Pichia and which was induced for expression. The expressed recombinant product was analyzed by SDS-PAGE and immunos- pot. Results The expanded AK cDNA was 1071 bp and the sequencing result showed 99. 5~ homolog compared with the sequence collected by GenBank （EU497674）. The relative molecular mass of the re- combinant protein was about 40 kDa showed by SDS-PAGE and Western blot showed it has a strong immunoreactivity. Conclusion The cDNA clone of AK gene from Metapenaeus ensis and the eukaryotic expressed recombinant protein were obtained, which established a basis for developing the diagnostic reagent of shrimp allergic disease.