转基因枸杞表达HIV-1壳体蛋白病毒样颗粒疫苗表达载体的构建

利用PCR技术扩增出人免疫缺陷病毒HIV-1MA₄-CA融合基因,将其克隆到pGEM-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为450bp,与已发表的HIV-1的全序列基因(AF324493)完全同源,编码一个含150个氨基酸残基的蛋白质。将该基因与分泌型表达载体pCAMBIA1305.2连接,同时将水稻中富含甘氨酸蛋白的信号肽序列(GRP)引入MA₄-CA融合基因,构建了含MA₄-CA基因的植物分泌表达载体pCAMBIA1305.2-MA₄-CA。

Construction of a Transgenic Lycium barbarum L. Recombinant Vector Expressing HIV-1 Capsid Protein Viral Like Particles

The capsid protein (CA) of HIV is an essential pr otein of the viral core, abundant in virion and forms its hydrophobic capsid. CA is highly conserved among different subtypes. CA existing alone in vivo can only form the column particles. However, once its N-terminal combines with at least four amino acid residues of the HIV matrix protein (MA), the CA will trans form to globosity, which leads to the enhancement of the original immunity of CA . The fused proteins MA_4-CA were expressed, and the spheral particles to enha nce the original immunity of CA were formed. Specific primers were used for ampl ification of the fused gene MA_4-CA encoding HIV-1 CA viral like particle (VLP). The fused gene MA_4-CA was ligated into the expression cassette pr esent in an intermediate plasmid pGEM-T. The fused gene was verified by the seq uencing identification, predicting its length was 450bp, homologous with HIV-1 complete sequence as published (AF324493). The fused gene MA_4-CA was then inserted in the plasmid vector pCAMBIA1305.2 with the rice glycine-rich protei n signal peptide sequence (GRP) after enzyme digestion. The secreting type plant expression vector pCAMBIA1305.2-MA_4-CA had been successfully construceted w ith GRP and MA_4-CA fused genes. The aim is to establish a botanical expre ssion system to produce high-safety HIV vaccine based on MA4-CA fused protein by the secretion and expression of the transgenic Lycium barbarum L.