pBacPAK8昆虫表达载体说明

pBacPAK8

编号 载体名称

北京华越洋生物KCVECT129 pBacPAK8

pBacPAK8载体基本信息

载体名称: pBacPAK8

质粒类型: 昆虫表达载体；杆状病毒表达载体

克隆方法: 限制酶，多克隆位点

启动子: Polyhedrin

载体大小: 5538 bp

5' 测序引物及序列: Bac1 Forward

3' 测序引物及序列: Bac2 Reverse

载体标签: 无标签

载体抗性: 氨苄青霉素

真核筛选标记: 无

克隆菌株: TOP10

宿主细胞（系）: 果蝇细胞系 IPLB-Sf21

备注: --

瞬表达/稳表达: 稳表达

组成型/诱导型: 组成型

病毒/非病毒: 杆状病毒

pBacPAK8载体质粒图谱和多克隆位点信息

pBacPAK8载体描述:

Available as part of the BacPAK Baculovirus Expression System (Cat. No. 631402). pBacPAK8 is a transfer vector designed for high-­‐level expression of a cloned gene driven by the strong AcMNPV polyhedrin promoter. Flanking AcMNPV sequences allow recombination with viral DNA to transfer the expression cassette to the polyhedrin locus of the viral DNA. The polyhedrin coding sequences have been replaced by a multiple cloning s ite w ith 18 u nique s ites t hat f acilitate t he i nsertion o f f oreign g enes i n t he c orrect orientation for expression. The Pac I site at the end of the MCS region provides translational s top c odons i n a ll t hree r eading f rames f or e xpression o f t runcated p roteins. pBacPAK8 has a pUC origin of replication, an M13 origin for single-­‐stranded DNA production, a nd a n a mpicillin r esistance g ene f or s election i n E. c oli.

1.杆状病毒表达系统简介

Baculovirus gene expression is a popular method for producing large quantities of recombinant proteins in insect host cells. In most cases, posttranslational processing of eukaryotic proteins expressed in insect cells is similar to protein processing in mammalian cells. As a result, insect cell-­‐processed proteins have comparable biological activities and immunological reactivities to proteins expressed in mammalian cells. Protein y ields f rom b aculovirus s ystems a re h igher, a nd c osts a re s ignificantly l ower t han in m ammalian e xpression s ystems. T he b aculovirus e xpression s ystem c an e xpress g enes from b acteria, v iruses, p lants, a nd m ammals a t l evels f rom 1–500 m g/liter; m ost p roteins are e xpressed i n t he 10–100 m g/liter r ange, a lthough m aking p redictions i s d ifficult.

The baculovirus most commonly used to express foreign proteins is Autographa californica nuclear polyhedrosis virus (AcMNPV). AcMNPV can be propagated in certain insect cell lines; the virus enters the cells and replication begins approximately 6 hours post-­‐infection (h.p.i.). At approximately 20–48 h.p.i., transcription of nearly all genes ceases. The viral polyhedrin and p10 genes, however, are transcribed at high rates. The polyhedrin p rotein i s e ssential f or p ropagation o f t he v irus i n i ts n atural h abitat; h owever, in cell culture, polyhedrin is not needed, and its coding sequence can be replaced with a sequence for a target protein. Hence, the powerful polyhedrin promoter can drive