【摘要】 目的构建转基因枸杞表达系统,并表达HIV壳体蛋白。方法以重组质粒pET-3a-MA4-CA为模板,PCR扩增MA4-CA基因,克隆至pGEM-T质粒中,转化DH5α,经双酶切后重组于pCAMBIA1305.2载体中,构成pCAMBIA1305.2-MA4-CA载体,经农杆菌介导叶盘转化法转化枸杞,培育转基因枸杞植株,进行芽、根诱导。结果在植物培养箱中,转基因植株成功地进行了分化及生根的诱导。PCR扩增分析表明转基因植株内存在目的基因,并与启动子p35S相连。ELISA及Westernblot实验证实在转基因枸杞叶子提取物中存在壳体蛋白。结论初步建立了表达具有免疫原性的HIV壳体蛋白的转基因枸杞表达系统。更多还原

【Abstract】 Objective To construct a transgenic Lycium barbarum L. expression system for HIV capsid protein（CA）.Methods The gene encoding MA₄-CA fusion protein was amplified by PCR using plasmid pET-3a-MA₄-CA as a template,then cloned into vector pGEM-T and transformed to E.coli DH5α.The recombinant plasmid was digested with BglⅡ and BstEⅡ,and the obtained MA₄-CA gene was inserted into secretory expression vector pCAMBIA1305.2.The constructed plasmid pCAMBIA1305.2-MA₄-CA was transferred into Lycium barbarum L. by Agrobacterium tumefaciens-mediated transformation,and the transgenic plant was cultivated for budding and rooting.Results Budding and rooting of the transformants was successfully induced in the growth chamber.PCR amplification of DNA from transgenic plant confirmed the presence of the fusion gene under the control of promoter p35S and the ligation between the MA₄-CA fusion gene and the promoter.ELISA and Western blot proved the presence of CA in transformed leaf extracts.Conclusion The findings indicated that the constructed transgenic Lycium barbarum L. expression system can be used to produce immunogenic CA.更多还原