摘要
将霍乱毒素B亚单位 (CTB)基因克隆到质粒pBin4 38中 ,分别构建了植物表达载体pBI CTB、pBI SPCTB和pBI CTBER。采用叶盘法分别转化烟草K32 6,各表达载体得到了一批转基因植株。转基因烟草的PCR和Southernblot分析表明CTB基因整合到了烟草基因组中。转基因植株的ELISA和Westernblot分析表明pBI SPCTB和pBI CTBER的转基因植株能有效表达CTB蛋白 ,分别占烟草叶片可溶总蛋白的 0 .0 95 %和 0 .115 % ,比pBI CTB转基因植株大约高 2 7倍。转基因烟草表达的CTB能够形成寡聚体或五聚体。
Three plant transformation vectors (pBI?CTB, pBI?SPCTB and pBI?CTBER) were constructed to express CTB under the control of the CaMV 35S promoter. Tobacco was transformed by co?cultivating leaf discs with Agrobacterium strains harboring pBI?CTB, pBI?SPCTB and pBI?CTBER respectively. The regenerated kanamycin?resistant transformants were analyzed by PCR, Southern blot, ELISA and Western blot. These results indicated the CTB gene integrated in the tobacco genomic DNA and was expressed efficiently. CTB levels in the pBI?SPCTB and pBI?CTBER lines were up to 950 μg and 1 150 μg per gram of total soluble tobacco leaf protein separately. It is up to 27 times more than that in the pBI?CTB lines. These results suggested that pBI?SPCTB and pBI?CTBER caused proper cellular compartmentation of the fusion protein, thus facilitating oligomerization.
出处
《生物技术通讯》
CAS
2000年第2期98-102,106,共6页
Letters in Biotechnology
