动物病原大肠杆菌K88的绿色荧光蛋白基因标记及稳定性的研究.pdf

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 9 K88 车建美 刘波 蓝江林 (, 350003) : 成功地将 gfp/ luxAB双标记基因整合到K88染色体上,得到绿色荧光蛋白基因标记的 肠杆菌K88 gfp/ lux,其 菌体和菌落形态与原始菌株 K88完全一致, 引入的新质粒不影响菌株的基本形态从含 gfp基因的质粒 DNA 和 K88 gfp/ lux 基因组 DNA上均可扩增出 小约 700 bp的 gfp基因片段 肠杆菌特异性基因检测结果表明,从 肠杆菌 K88和 K88 gfp/ lux基因组 DNA 上均扩增出 小约 260 bp的 肠杆菌特异性基因片段,说明 gfp基因标记后的菌株均为 肠杆菌在相同的 培养条件下, K88 gfp/ lux和 K88的生长曲线的变化趋势基本相同通过检测肠毒性基因( estA)发现, 从 肠杆菌 K88和K88 gfp/ lux基因组 DNA上均扩增出 小约 158 bp的肠毒性基因片段,说明 gfp基因标记后的菌株在肠毒性方面未发生变化在 无选择压力条件下将 K88 gfp/ lux菌株每隔 12 h连续转接 10次后, 所有菌落均保持着均匀并且强烈的绿色荧光,说明标记基 因在 K88 gfp/ lux中的表达稳定性很高K88 gfp/ lux和 K88在中性偏酸性的环境中生长较好, 当初始 pH 值偏碱性时, 生长 较差 : 肠杆菌K88 绿色荧光蛋白 gfp/ luxAB基因 Transform ation and Expression of gfp G ene into E scherich coli K88 he Jianmei L iu Bo Lan Jianglin (Agricultural B ioresource Research Institute, Fuj ian A cademy of Agricultural Sciences, Fuzhou 350003) Abstrac:t TheE scherich coli K88 strain w as chromosomally tagged w ith themarker genes gfp and luxAB by electroporation. The transformantwas screened for the green fluorescence by fluorescence stereom icroscopy. M orphologically, the cells of engineered strain K88 gfp/ lux were smi ilar to w ild type as determ ined by laser scanning confocalm icroscopy. A 700 bp fragmentof the gfp genew as am plified from the genome DNA of the GFPtagged strains, and a 260 bp fragmentw as amplified from the genome DNA ofK88 andK88 gfp/ lux w hich showed that theGFPtagged strainw asE. coli. The green fluorescence could be keptuntilmore than 10 tmi es of subcul tures. The tested of diseasecausing virulent gene ( estA gene) of K88 andK88 gfp/ lux show ed that a 158 bp fragment could be ampli fied in them. The insertion of gfp gene did not change the expression of the v