試劑中英文名稱(chēng):pETBlue-2 Vector 大腸桿菌表達(dá)載體
儲(chǔ)存:-20℃干燥避光保存至少1年,冰袋運(yùn)輸。
載體標(biāo)簽: C-HSV,C-6×His
一、背景描述:(pETBlue-2 Vector 大腸桿菌表達(dá)載體)
The pETBlue vectors are designed to identify recombinants by traditional blue/white screen-ing while also allowing T7lac promoter based expression of target genes.
Screening is independent of expression because the T7lac expression promoter is in an opposed orientation relative to the E.coli promoter that mediates blue/white screening.
pETBlue-2 defines the open reading frame and in-serts must be cloned in-frame if expression is desired.
The vector features an expanded multiplecloning site (MCS) and optional C-terminal HSV•Tag and His•Tag sequences.
The cloning/expres-sion region of the coding strand transcribed by T7 RNA polymerase is shown below.
The sequence is numbered from the first base of the T7 promoter sequence.
Unique sites are shown on the circlemap.
The f1origin in pETBlue-2 is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand.
Therefore, single stranded sequencing should be performed using the pETBlueDOWN prime.
目標(biāo)基因以反義方向插入lacZα肽編碼區(qū),實(shí)現(xiàn)藍(lán)白篩選;而T7轉(zhuǎn)錄/翻譯信號(hào)位于基因上游,確保表達(dá)時(shí)以正義方向工作。
T7啟動(dòng)子不被大腸桿菌RNA聚合酶識(shí)別,僅由T7 RNA聚合酶(由DE3溶源噬菌體提供)特異性激活,實(shí)現(xiàn)本底泄漏低、誘導(dǎo)后高表達(dá)。
利用T7基因10的翻譯起始信號(hào)(RBS),提升目標(biāo)蛋白產(chǎn)量。
二、應(yīng)用前景
重組蛋白表達(dá):廣泛用于克隆、表達(dá)與純化外源蛋白,尤其適合需要可溶性表達(dá)的課題。
蛋白互作研究:融合標(biāo)簽支持Pull-down、Co-IP等互作實(shí)驗(yàn)。
載體設(shè)計(jì)理念(如高效啟動(dòng)子、標(biāo)簽系統(tǒng))已被用于疫苗抗原表達(dá)平臺(tái),展示其在生物制劑生產(chǎn)的潛力。
藍(lán)白篩系統(tǒng)適用于大規(guī)模重組庫(kù)篩選,為突變體文庫(kù)構(gòu)建提供便利。
藍(lán)白篩選手動(dòng)直觀,大幅減少假陽(yáng)性;高拷貝數(shù)pUC復(fù)制起點(diǎn)(>500拷貝/細(xì)胞)提升質(zhì)粒產(chǎn)量。
IPTG誘導(dǎo)濃度可調(diào),適用于毒性蛋白或優(yōu)化表達(dá)條件。
雙標(biāo)簽(6×His/HSV•Tag)支持多種純化策略(如鎳柱、抗體親和層析)和檢測(cè)方法。
與pET系統(tǒng)宿主(如BL21系列)高度匹配,Lon/OmpT蛋白酶缺陷型菌株減少蛋白降解。
質(zhì)粒以?xún)龈煞坌问教峁?℃避光保存,穩(wěn)定性好;配套詳細(xì)操作流程(溶解、轉(zhuǎn)化、保存)。
用于科研相關(guān)領(lǐng)域,不能用于人體實(shí)驗(yàn)!
西安強(qiáng)化生物提供的相關(guān)系列:
Safranine O, High Purity Grade
5-TAMRA azide 5-羧基四甲基羅丹明疊氮化物
6-ROX azide 6-ROX 6-羧基-X-羅丹明疊氮化物
AMCA, succinimidyl ester AMCA 琥珀酰亞胺酯
Plant Preservative Mixture (PPM) 植物組培抗菌劑
Plant Preservative Mixture (PPM) 植物組培抗菌劑