The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae s...The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.展开更多
PD-1(Programmed cell death 1)是一种抑制性的受体,是免疫球蛋白超家族的成员.大量研究证明PD-1能被诱导而在活化的T淋巴细胞、B淋巴细胞、NKT细胞和单核细胞上表达,从而在慢性感染的病因学中起着重要作用.树鼩(Tupaiabelangeri)做为...PD-1(Programmed cell death 1)是一种抑制性的受体,是免疫球蛋白超家族的成员.大量研究证明PD-1能被诱导而在活化的T淋巴细胞、B淋巴细胞、NKT细胞和单核细胞上表达,从而在慢性感染的病因学中起着重要作用.树鼩(Tupaiabelangeri)做为一个理想的模型可被应用于许多人类感染性疾病如乙型病毒性肝炎.为了充分利用树鼩对于感染性疾病的宿主免疫应答模型,我们分离出树鼩PD-1基因.利用迅速扩增cDNA末端PCR(RACE-PCR)技术,从树鼩脾组织中克隆了PD-1基因的全长cDNA序列.序列分析显示树鼩PD-1 cDNA的开放阅读框编码一个由242个氨基酸组成的跨膜蛋白,并且和人类、灵长类和啮齿类中的同源基因有高度相似性.组织分布分析表明在所检测的几种组织中PD-1基因只在脾中表达.此外,淋巴细胞刺激实验显示,利用PMA和ionomycin刺激新鲜分离的树鼩外周血单核细胞(PBMCs)能够诱导PD-1 mRNA水平上的表达.我们的结果为将来进一步探讨树鼩的免疫功能提供了良好的基础.展开更多
文摘The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.
