Study On Gene Cloning And Expression Of RIP From The Bouganinvillea.glabra

Many plants contain proteins with antiviral activity. Some of these proteins belong to the family of ribosome-inactivating proteins(RIPs), which direcly inhibit the protein synthesis of ribosomes. RIPs can be divided in two grous on the basis of their structure, type-I RIPs and type-II RIPs. Bouganin is a type-I RIP that purified from Bougainvilled spectabilis Willd, it was found that Bouganin is the least toxic RIP known.The Bouganin sequence was cloned from the leaves of seeds of Bougainvillea glabra by PCR. Not I site was introduced at the upstream primer whereas a stop codon and Xho I site were added at the downstream. The nucleotide sequences of the target DNA amplification fragment were sequenced after T-A cloning. The Bouganin gene is 750 bp and encodes a protein of 250 amino-acid residues. In comparison with the reported Bouganin sequence from the B.spectabillis Willd, the nucleotide sequenceomology of the cloned Bouganin gene was 83%. And in the protein residues the 229th amino-acid is a stop codon. The fragments digested with restriction endonucleases Xho I and Not I were subsequently cloned into the vector pET-28a. E. coli BL21 (DE3) transformed with the expression plasmid was grown in LB at 37℃to an optical density of 0.6. Expression was induced by adding IPTG to a final concentration of 0.5 mmol/L and incubation with shaking at 28℃for 6 h, this is the best induced condition for Bouganin gene expression. The product was identified by 12% SDS-PAGE. The results showed that the recombinant product is about 29 kDa and it mainly existed in inclusion body. The recombinant protein in inclusion body was denatured with 8mo1/L urea and was renatured by dialysis. The renatured and purified proteins showed a single band on 12%SDS-PAGE. The cytotoxicity of Bouganin was evaluated by the MTT assay in HepG2 cells following fluid-phase endocytosis. The results of MTT assay showed the Bouganin possessed toxicity to HepG2 cells.In this article, we report that Bouganin gene has been successfully cloned from the leaf of Bougainvillea glabra (Sanderiana) from XiaMen and expressed in E. coli. The deeply research of Bouganin gene can generates a prospect for the application in agriculture and the...