The transfermants with highest resistance to g418 ( 4 g / l in ypd ) were screened to study their expression level in 150 ml shaking flask . having been induced with methanol for 36 hours , the target enzyme could be examined in the supernatant by measuring amidolytic activity 首次将大连蛇岛赅蛇毒类凝血酶成熟基回克隆到表达载体ppicgk中，经电激转化至毕氏酵母菌株gs15中，再经甲醇诱导，在150ml摇瓶畔1获得细胞外分泌表达产物。
Hegf gene with his - tag at the end , which was derived from pet22 - egf , was in - frame fused to the carboxy - terminal of polyhedrin ( ph ) gene , which included the amino - terminal 116aa coding region . the polyhedrin - egf fusion gene ( named ph - egf ) was then cut out with ecorv and ecori , and was cloned between ecorv and ecori sites of pbacpak . 8 ( the result plasmid was named pbacph - egf and the ph - egf fusing gene was right under the control of ph promoter ) . the pbacph - egf structure was verified with restriction enzymes digestion and pcr 从pet22 - egf质粒中分离出末端带his - tag的egf基因，对位融合于多角体蛋白n端116个氨基酸基因序列的下游(命名为ph - egf ) ，并在两段基因间设计了凝血酶xa蛋白酶切位点，经过酶切、测序等鉴定正确后，克隆至pbacpak8中，使ph - egf融合基因置于多角体蛋白( polyhedrin ， ph )基因启动子控制之下，构建成重组转移载体pbacph - egf 。
According to the purity and the activity recovery , the recombinant enzyme was well purified by both of these two separation combinations . like natural gloshedobin , the recombinant enzyme exhibited strong esterase activity using tripeptide p - nitroanilide derivatives as substrate , but hydrolyzed n a - p - tosyl - l - arginine methyl ester ( tame ) or n a - benzoyl - l - arginine ethyl ester ( baee ) very weakly 与天然蛇毒类凝血酶一致，当用三肽p山itroanilide衍生物作为底物时，分泌表达的重组大连蛇岛蝗蛇毒类凝血酶具有较强的生色底物活性，但用精氨酸甲酯如tame （ na厂tosyl l ar 。
Both the mature genes of gloshedobin and gussurobin were cloned into the vector pet - 32a ( + ) , strain bl21 ( de3 ) , to study their expression in prokaryotic cell . the gene was expressed under t7 promoter with a fusion protein partner of thx . tag and a 6x his . tag at its n - terminal . having been induced by iptg for 4 hours , the recombinant enzyme was examined in the cytoplasm by sds - page analysis 将大连蛇岛蝮蛇和长白山白眉蝮蛇毒类凝血酶基因分别克隆到大肠杆菌表达载体pet - 32 ( a ) +中，在t7启动子下表达出融合蛋白，融合部分为硫氧还蛋白，位于类凝血酶基因上游，并在其n端带6xhistag标签以利于表达产物的分离纯化，经热激转化至宿主菌bl21 ( de3 )中， iptg诱导斗小时后收获菌体。
Its amino acid sequence exhibits significant homology with those of other thrombin - like snake venom enzymes . based on the homology , the catalytic residues and disulfide bridges of gloshedobin were deduced to be as follows : his43 , asp88 , ser182 and asp176 ; and disulfide bridges , cys7 - 141 , cys 28 - 44 , cys 76 - 234 , cys120 - 188 , cys152 - 167 and cys178 - 213 以同样的方法，得到长白山白眉蝮蛇( gloydiusussuriensis )毒类凝血酶基因，并比较了它与大连蛇岛蝮蛇类凝血酶基因序列的差别，发现两者的同源性高达98 ，只有三个氨基酸不同： gln ~ ( 66 )
