vector nti 載體分析

1、Vector NTI 載體（載體（vector） ，指在基因工程重組，指在基因工程重組DNA技術(shù)中將技術(shù)中將DNA片片段（目的基因）轉(zhuǎn)移至受體細(xì)胞的一種能自我復(fù)制的段（目的基因）轉(zhuǎn)移至受體細(xì)胞的一種能自我復(fù)制的DNA分子。分子。載體必備條件含有復(fù)制起始位點(diǎn)，能夠獨(dú)立復(fù)制,在宿主細(xì)胞中能保存下來(lái)并能大量復(fù)制。有多個(gè)限制酶切點(diǎn)，而且每種酶的切點(diǎn)最好只有一個(gè)。有一定的標(biāo)記基因，便于進(jìn)行篩選。三種最常用的載體是細(xì)菌質(zhì)粒、噬菌體和動(dòng)植物病毒。pBR322是一個(gè)人工構(gòu)建的重要質(zhì)粒，有萬(wàn)能質(zhì)粒之稱。它是由pSF2124、pMB1及pSC101三個(gè)親本質(zhì)粒經(jīng)復(fù)雜的重組過(guò)程構(gòu)建而成的。p BR 322Plasm

2、id研究者Bolivar和Rogigerus實(shí)驗(yàn)編號(hào)如何讀懂質(zhì)粒圖譜載體必備條件含有復(fù)制起始位點(diǎn)，能夠獨(dú)立復(fù)制,在宿主細(xì)胞中能保存下來(lái)并能大量復(fù)制。有多個(gè)限制酶切點(diǎn)，而且每種酶的切點(diǎn)最好只有一個(gè)。有一定的標(biāo)記基因，便于進(jìn)行篩選。Ori的位置，了解質(zhì)粒的類(lèi)型（原核/真核/穿梭質(zhì)粒）看多克隆位點(diǎn)（MCS）篩選標(biāo)記載體必備條件含有復(fù)制起始位點(diǎn)，能夠獨(dú)立復(fù)制,在宿主細(xì)胞中能保存下來(lái)并能大量復(fù)制。有多個(gè)限制酶切點(diǎn)，而且每種酶的切點(diǎn)最好只有一個(gè)。有一定的標(biāo)記基因，便于進(jìn)行篩選。Ori的位置，了解質(zhì)粒的類(lèi)型（原核/真核/穿梭質(zhì)粒）看多克隆位點(diǎn)（MCS）篩選標(biāo)記表達(dá)系統(tǒng)元件pBR322質(zhì)粒載體的構(gòu)建過(guò)程可簡(jiǎn)單

3、地概括如下：1、由于pBR322質(zhì)粒的親本之一是pMB1質(zhì)粒，故首先以pMB1為基礎(chǔ)，引入Rldrd19質(zhì)粒的Tn3易位子，得到13.3kb的pMB3質(zhì)粒；2、pMB3的分子量顯然使得它不適合作為載體，所以要通過(guò)在EcoRI活性條件下的消化讓它大部分的無(wú)用片段失去，留下來(lái)的小片段的DNA的黏性末端連接起來(lái)后就形成了pMB8質(zhì)粒（2.6kb）；3、此時(shí)，另外一種pSC101質(zhì)粒在EcoRI活性條件下消化產(chǎn)生了含有tetr抗性的DNA片斷，這個(gè)片斷和pMB8整合在一起就形成了pMB9質(zhì)粒（5.3kb）。此時(shí)pMB9為ampstetr表型；4、pMB9已經(jīng)初步具備載體的功能，為了讓它更加完善，我們要

4、讓它具備amprtetr性能，即在pMB9上引入pSF2124的Tn3易位子，但Tn3可以來(lái)也可以走，為了留住它，我們切去了Tn3中表達(dá)轉(zhuǎn)位酶的基因，形成了pBR313質(zhì)粒。此后我們兵分兩路：一路把pBR313的PstI位點(diǎn)除去，使之成為ampstetr表型的pBR318質(zhì)粒；5、另一路把pBR313的EcoRII的位點(diǎn)切除，使之成為amprtets表形的pBR320質(zhì)粒。由此我們的到了兩種功能互補(bǔ)的質(zhì)粒，這樣我們只要將他們雜交就可以得到一種接近全能的載體質(zhì)粒了，這就是pBR322 。pBR322 is a plasmid and was one of the first widely use

5、d E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after the postdoctoral researchers who constructed it. The p stands for plasmid, and BR for Bolivar and Rodriguez.pBR322 is 4361 base pairs 1 in length and harbo

6、rs the origin of replication of plasmid pMB1, a close relative of the plasmid ColE1. pBR322 also contains the ampR gene, encoding the ampicillin resistance protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101), and the rop gene, enco

7、ding a restrictor of plasmid copy number. The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tetR gene. There are 6 key restriction site

8、s inside the ampR gene.2The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet gene. The ampicillin resistance gene is penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcript