摘要
利用随机克隆的枯草杆菌启动子-信号序列构建茅孢杆菌分泌载体pUS186。用限制酶将切除了信号序列的猪生长激素cDNA从质粒pLY3-PGH 604切下,亚克隆至pUS186,并在该cDNA的下游接上地衣杆菌α-淀粉酶基因的转录终止子,构建猪生长激素表达质粒pSGH 1864,将此质粒转化蛋白酶双缺陷的枯草杆菌DB104及短小茅孢杆菌289。SDS-聚丙烯酰胺凝胶电泳检出在发酵上清液中多出一条22kD的蛋白带,抗猪生长激素血清免疫印迹法证明这一蛋白带具有免疫活性,表明猪生长激素cDNA已在枯草杆菌及短小茅抱杆菌中表达。
Secretive expression vector pUS186 was constructed using the promotersignal sequence coding region cloned from Bacillus subtilis chromosome. The 604bp porcine growth hormone (PGH) cDNA without signal sequence was excised from plasmid pLY3-PGH604 and ligated to secretion vector pUS186 and a 0.4kb fragment containing the terminator of α-amylase gene from B.lichniformis was inserted downstream of the PGH cDNA. The resulted plasmid was designated as pSGH1864 and transformed into double protease mutant of Bacillus sabtilis DB104 and Bacillus pumilus 289, SDS-PAGE and Western blot with anti-PGH serum show that the PGH cDNA was expressed both in B.subtilis and B.pumilus.
基金
国家"七五"重点攻关项目(75-71-01-02)
广东省青年科学基金
关键词
猪
生长激素
基因表达
枯草杆菌
Porcine growth hormone
Gene expression
B.subtilis
B.pumilus
