毕赤酵母中抗癌镇痛肽肺靶向融合体表达载体——穿梭质粒pPIC9K-RGD-4C-His Tag-AGAP的构建

采用PCR的方法对AGAP(anti-cancer analgesic peptide,抗癌镇痛肽)的N端进行了基因改造,保留了Kex2初步切割信号序列,去除了Ste13的切割序列,加入了对蝎毒蛋白抗癌镇痛活性无影响的氨基酸作为linker。通过构建辅助质粒pPIC6K,避免了kan基因内部的Xho I酶切位点对基因克隆的影响,构建了中介质粒pPIC6K-RGD-4C-His Tag-AGAP,该质粒经BamH I和EcoR I双酶切取得所需DNA片段,与同样经过双酶切的质粒pPIC9K进行连接,最终成功构建了酵母表达质粒pPIC9K-RGD-4C-His Tag-AGAP,测序结果表明,抗癌镇痛肽肺靶向融合体基因已成功插入到酵母表达载体pPIC9K中。

Expression vector of a lung targeted fusion protein AGAP in Pichia pastoris, construction of plasmid pPIC9K-RGD-4C-His Tag-AGAP

N terminal of AGAP was arranged through PCR.KEX2 cutting site of signal sequences was retained.Ste13 cutting was removed.A linker was added without influencing the activity of AGAP.The gene of targeting peptides was inserted into 5′ end of the gene of anti-cancer analgesic peptide by PCR.Through constructing auxiliary plasmid pPIC6K,the influence of Xho I site in gene kan was avoided,and medium plasmid pPIC6K-RGD-4-His Tag-AGAP was constructed.Through double enzymes digestion,interested DNA fragment was cut off,and then it was linked into plasmid pPIC9K which was also digested by the same two enzymes.Sequencing results showed that lung cancer targeted AGAP was already inserted into plasmids pPIC9K successfully.