产品介绍
pAcGP67A昆虫分泌质粒
基本信息
启动子: Polyhedrin
复制子: ColE1 ori
终止子: polyhedrin 3
质粒分类: 昆虫细胞载体;杆状病毒组成型分泌表达质粒
质粒大小: 9761bp
质粒标签: N-GP67 signal
原核抗性: 氨苄青霉素Ampicillin
克隆菌株: DH5α
培养条件: 37℃,有氧,LB
表达宿主: 昆虫细胞
诱导方式: 组成性表达,无需诱导
5'测序引物: Polyhedrin-F:AAATGATAACCATCTCGC
3'测序引物: Polyhedrin-R:GTCCAAGTTTCCCTG
备注: 含有GP67分泌信号肽
质粒简介
The acidic glycoprotein gp67 (syn.: gp64) is the most abundant envelope surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV baculovirus), and is essential for the entry of baculovirus particles into susceptible insect cells. Since large amounts of this protein are secreted and anchored to the virus peplomer, its gene contains one of the most effective baculovirus-encoded signal sequences for protein secretion. Therefore, we have constructed baculovirus transfer vectors that contain the gp67 signal sequence in front of a multiple cloning site. A gene of choice can be inserted in one of these cloning sites and the protein of interest will be expressed as a gp67 signal peptide fusion protein under the control of the strong baculovirus polyhedrin promoter. This strategy allows the forced secretion of otherwise non-secreted recombinant proteins which may be easily purified when serum-free insect culture medium, BD BaculoGold Max-XP Insect Cell Medium is used. The transfer vector(s) should be preferentially used in conjunction with BD BaculoGold DNA .
质粒图谱
pAcGP67A昆虫分泌质粒使用说明:
1、收到质粒干粉后请先5000rpm离心1min,再加入20μl无菌水溶解质粒,室温放置1min;
2、从-80℃冰箱中取出相应的感受态,置于冰盒上解冻,并做好标记;
3、取2μl质粒加至100μl感受态中,冰浴30min;
4、42℃热激90s,再冰浴2min;
5、加入900μl无抗的LB液体培养基,180rpm震荡培养45min;
6、6000rpm离心5min,仅留100ul上清混匀菌体沉淀;
7、混匀后的菌液加至对应抗性的LB平板上,倒入适量玻璃珠,涂匀液体;
8、将平板正向培养1h,再倒置培养12h~16h;
9、挑取单克隆菌落至对应抗性的LB液体培养基中,震荡培养12h~16h,根据实验需要提取质粒。
pAcGP67A昆虫分泌质粒注意事项:
1、如果您收到的是甘油菌种,请先四区划线,挑取单克隆培养。
2、如果第二天转化平板长的过多,请将质粒按比例稀释后再转化。