丝瓜籽核糖体失活蛋白luffin-a基因的克隆和表达

Cloning and expression of luffin-a gene from the seeds of Luffa cylindrical

目的:从丝瓜籽中克隆HIV整合酶抑制剂luffin-a基因,并构建其表达载体。方法:根据报道的cDNA序列,用RT-PCR的方法从未成熟的丝瓜籽中克隆luffin-a基因,T-A克隆后鉴定核苷酸序列。构建表达载体pET-44a(+)-luffin-a,转化大肠杆菌BL21(DE3),并经IPTG诱导表达。用透析法对包含体复性;SDS-PAGE鉴定luffin-a表达产物,用MTT法分析其生物学活性。结果:克隆的luffin-a基因与国外报道的核苷酸序列同源性为99.73%,其所编码的氨基酸序列完全一致。经SDS-PAGE分析:luffin-a主要存在于包含体中。MTT法测定:复性后蛋白具有与蓖麻毒素A链相似的细胞毒性。结论:本研究成功地构建了luffin-a的表达系统,所表达蛋白经复性后具有生物学活性。

<Abstrcat>Objective: To clone luffin-a cDNA from the seeds of Luffa cylindrical,and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a(+) in E.coli. Methods: The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR.The target DNA fragments were sequenced after T-A cloning.The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a(+).Luffin-a was expressed in E.coli by addition of IPTG into final concentration 1.0 mmol/L.The recombinant luffin-a was identified by SDS-PAGE.The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis. Results: In comparison with the reported luffin-a,the homology of nucleotide sequence ofthe cloned luffin-a gene was 99.73%,while their amino acid sequences were identical.The solubility of recombinant protein was analyzed by SDS-PAGE,and the luffin-a was mainly produced in inclusion bodies.The recombinant luffin-a,renatured by dialysis of the denatured products,showed a similar cytotoxicity to ricinA chain. Conclusion: The cDNA of luffin-a has been successfully cloned.The recombinant luffin-a protein expressed by E.coli is bioactive.