中国对虾Rab7全长cDNA的克隆及原核表达

cDNA cloning and prokaryotic expression of Rab7 from shrimp Fenneropenaeus chinensis

本研究采用RACE方法克隆了中国对虾Fenneropenaeus chinensis Rab 7基因(fcrab7)全长cDNA(GenBank:JF742050)。生物信息学分析显示,fcrab7的开放阅读框615bp,编码205个氨基酸,分子量23.2kD。FcRab7的氨基酸序列具有Rab蛋白家族的共同特征,与凡纳滨对虾和斑节对虾同源蛋白的同源性为100%。构建重组表达载体pBAD/gIIIA-fcrab7转化到大肠杆菌E.co-li进行诱导表达,诱导产物经SDS-PAGE检测和Westernblot验证。本研究结果为进一步研究WSSV与FcRab7的相互作用提供了基础。

Rab proteins are the largest subfamily in small GTP-binding proteins, which play important roles in vesicle formation, transportation, adhesion, anchoring,and fusion. As a member of this family, Rab7 protein is in charge of the delivery of internalised materials into degradative compartments and the acquisition of lysosomal hydrolases. Evidence from the previous studies showed that VP28 of white spot syndrome virus （WSSV） can bind with Rab7 in Penaeus monodon （PmRabT）. By rapid amplification of cDNA ends （RACE） method, the fulllength gene of Fenneropenaeus chinensis Rab7 gene （fcrab7） was obtained. The total cDNA consists of a 615bp open reading frame （ORF）, which encoded 205 amino acids with a predicted molecular mass of 23.2 kDa . The deduced amino acid sequence contained many conserved motifs and regions, and the entire protein has 100 % homology with Rab7 of Litopenaeus vannamei and P. monodon. The recombined vector of pBAD/gIIIA-fcrab7 was constructed and transformed into E. coll. The fusion protein was successfully expressed by induction, which was tested by SDS-PAGE and Western blot analysis. These results laid a basis for further studies on interaction between WSSV and FcRabT.