Study Of Gene Cloning, Expression Of Ribosome-inactivating Protein From Atriplex Patens In Xinjiang

Many ribosome-inactivating proteins (RIPs) were identified in high plant since the first Ricin was isolated from Ricinus communis, and also found in fungus and bacteria. RIPs are a group of proteins that share the property of damaging ribosomes in an irreversible manner, acting catalytically, i.e. enzymatically. The mechanism of the ribosomal damage was discovered by Endo et al. who found that ricin cleaved the glycosidic bond of a single adenine residue (A4324 in rat liver rRNA). This residue is adjacent to the site of cleavage of rRNA by a-sarcin, in a tetranucleotide GA4324GA in a highly conserved loop at the top of a stem, for this called a-sarcin/ricin loop. This observation was extended to other RIPs, which were officially classified as rRNA N-glycosidases. RIPs are divided into two broad groups, type 1 RIPs, consisting of a single peptidic chain of 30 kDa, approximately, and type 2 RIPs which consist of an enzymatically active a chain similar to type 1 RIPs, linked to a slightly larger (35 kDa, approx) B chain, which has the properties of a lectin with specificity for sugars with the galactose structure. Along with the studying of RIPs, its practical application has also received the widespread attention. At present, it has involved in anti-viral, anti-fungus, anti-insect, anti-tumor and AIDS aspects and so on treatment.Based on already the literature research foundation which has, we did the experiments in the following four aspects on RIP from Atriplex patens:At first, the conservative region of the variable rip gene sequences published in GenBank were analyzed, and six primers (4 upstream, 2 downstream) were designed for amplifying core fragment of RIP according to these conservative region. Total RNA was isolated from the plant tissue and cDNAs were synthesized by reverse transcription. The different combinations of these primers were used to amplify the core fragment of RIP. The core fragments were cloned into pMD18-T cloning vector; positive clones were checked with restriction enzyme digestions and further identified with sequence analysis. With a set of primers designed according to the sequence of the cloned core fragments, the 3′ends of the cDNAs were obtained by 3′rapid amplification of cDNA ends (RACE). Combining the sequences of the 3′ends, we use anthor core primer from 5′ends to amplification of the open reading frame (ORF) of the Aprip. PCR product was cloned into pMD18-T cloning vector and sequenced. The result shows that ORF of the Aprip was obtained. And then it was registered in GenBank (GI: DQ991968) and named as Aprip. Sequence analysis reveals that the cloned Aprip fragment of Atriplex patens contains partial rip coding region with 82%,55% and 41% identity comparing with Chenopodium album, pokeweed antiviral protein and Trichosanthes kirilowii trichosanthin, respectively. The analysis suggests that ApRIP belongs to typeⅠRIP.Secondly, to express Aprip in vitro for further study of its characterization, Aprip gene was cloned into plasmid pMAL-p2x for expression Aprip gene in prokaryotic. SDS-PAGE analysis show that ApRIP was expressed in E.coli, and ApRIP was purified by the Amylose Resin Column.Thirdly, to expression of Aprip from Atriplex patens in eukaryote cells, Aprip was cloned into pcDNA3.1 to form pcDNA3.1-Aprip DNA vaccine. Hydrodynamic-based Transfection Method showed the transcription of pcDNA3.1-Aprip in the liver in mice, and ELASA and Western blot showed that the high and strong specific antibody was obtained.Finally, to further study the function of ApRIP, in one hand, Aprip was cloned into pET30a to form pET30a-Aprip. In order to compare the effect of different vector fusion protein on ApRIP, pET30a-Aprip has proved to translate to mRNA by RT-PCR, and there has been obvious inhibitory effect on E.coli, but it isn't examined the corresponding protein banding by SDS-PAGE, we guess that ApRIP expressed in E.coli was very low. In the other hand, pcDNA3.1-Aprip was constructed to inject in the mouse muscle cell, and we observe mouse's pate hair to start to fall off, their feed also has reduction compared to the control group.