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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (29): 64-71.

所属专题： 生物技术

• 水产 渔业 • 上一篇    下一篇

三角帆蚌Rab3 cDNA全长克隆及其原核表达

许宝红 肖调义 刘巧林 苏建明 陈开健 刘敏 周伟   

  • 收稿日期:2011-07-18 修回日期:2011-08-19 出版日期:2011-11-15 发布日期:2011-11-15
  • 基金资助:

    三角帆蚌丝氨酸蛋白酶基因的克隆、表达调控与功能研究

Full-length cDNA cloning and prokaryotic expression of Rab3 gene from Hyriopsis cumingii

  • Received:2011-07-18 Revised:2011-08-19 Online:2011-11-15 Published:2011-11-15

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摘要：

根据三角帆蚌消减杂交cDNA文库获得的EST序列，运用cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）技术获得三角帆蚌Rab3基因的全长cDNA序列（GenBank登录号为HQ190954）。序列全长1707 bp，开放阅读框666 bp，编码221个氨基酸，5’端非编码区为158 bp，3’端非编码区为883 bp。软件推测其编码蛋白相对分子量为24.92 KDa。NCBI Blastp进行氨基酸序列同源性分析表明，与居蟹皮海绵（Suberites domuncula）的Rab-3like（SdRab3）氨基酸相似性最高，为64%，构建进化树的结论也是先与居蟹皮海绵的Rab3-like（SdRab3）聚为一支。根据获得的Rab3基因cDNA全长序列，设计特异性引物扩增获得完整开放阅读框序列，并将已克隆到的三角帆蚌Rab3开放阅读框定向插入原核表达载体PQE-His质粒中，转化大肠杆菌BL21（DE3）。在30℃，5 h，1 mM IPTG的诱导条件下，Rab3蛋白得到高效表达，重组菌体经裂解和SDS-PAGE电泳分析，发现一条26 kDa左右特异的蛋白表达条带，其表达量约占菌体总蛋白30%。Western-blot检测表明，在健康组和病变三角帆蚌肝脏中，Rab3蛋白与β-actin的表达量比值分别为0.438和0.580。这一结果说明，Rab3基因在三角帆蚌经病原菌侵染后，表达量上调，且已有研究表明Rab3有通过调节中性粒细胞的胞吐作用来抵抗细菌侵入、防止机体感染的功能，从而初步推测三角帆蚌Rab3蛋白可能与三角帆蚌瘟病有关。

关键词: 双向电泳, 双向电泳

Abstract:

According to the Hyriopsis cumingii (H. cumingii) expressed sequence tags (EST) obtained by constructing subtractive hybridization cDNA library, the gene full-length cDNA sequence of Rab3 from H. cumingii was cloned by RACE-PCR technique(GenBank accession number: HQ190954).. The results showed that the length of Rab3 gene cDNA sequence was 1 707bp, containing a complete open reading frame 666bp, encoding a peptide of 221 amino acid residues(aa), flanked by a 158 bp of 5’untranslated region(UTR)and a 883 bp of 3’-UTR. The deduced molecular weight of Rab3 is about 24.92KDa. Amino acid sequence analysis shows that, Suberites domuncula Rab-3like and Hyriopsis cumingii Rab3 is the highest, about 64%, evolutionary tree conclusion is the same with that result. The Rab3 positives was therefore designaged HcRab3(GenBank accession number: HQ190954).The result of analysis with NCBI Blastp , which beween HcRab3 and SdRab3, which shares a positives of 64%.Induced with 30℃, 5h, 1mM IPTG, the HcRab3 was expressed at high levels, electrophoretic analysis by SDS-PAGE revealed that 26kD Protein expression band was obtained. After Western-blot in healthy and deseased Hyriopsis cumingii lever protein, the expression ratio of Rab3 protein and beta actin is 0.438 and 0.580. Rab3 can regulating exocytosis of body neutrophils, it is preliminarily presumed that the HcRab3 maybe related to Hyriopsis cumingii plague.