慢病毒載體系統(tǒng)是一種能非常高效的把外源基因穩(wěn)定整合到哺乳動(dòng)物細(xì)胞中的載體工具。除了常規(guī)質(zhì)粒轉(zhuǎn)染外，目前該系統(tǒng)也是把外源基因轉(zhuǎn)入哺乳動(dòng)物細(xì)胞的最常用方法之一。由于具有目的基因和啟動(dòng)子選擇的靈活性以及轉(zhuǎn)染細(xì)胞類型的廣泛性兩大特點(diǎn)，使得慢病毒載體系統(tǒng)成為倍受歡迎的外源基因表達(dá)系統(tǒng)。

慢病毒載體來源于人類免疫缺陷病毒HIV，屬于逆轉(zhuǎn)錄病毒家族。野生型慢病毒基因組是線性雙正鏈RNA。

慢病毒重組載體構(gòu)建完成后與輔助質(zhì)粒一起轉(zhuǎn)染進(jìn)入包裝細(xì)胞。在包裝細(xì)胞中，位于兩個(gè)長(zhǎng)末端重復(fù)序列（LTR）之間的DNA片段會(huì)被轉(zhuǎn)錄成RNA，由輔助質(zhì)粒表達(dá)的病毒蛋白將其包裝形成病毒顆粒。包裝后的活體病毒將會(huì)被釋放到上清液中，可以直接收集或進(jìn)一步濃縮病毒轉(zhuǎn)染靶細(xì)胞。

當(dāng)病毒轉(zhuǎn)導(dǎo)靶細(xì)胞時(shí)，釋放到宿主細(xì)胞中的病毒RNA借助逆轉(zhuǎn)錄酶逆轉(zhuǎn)錄成雙鏈DNA，然后隨機(jī)整合進(jìn)宿主細(xì)胞的基因組中。在病毒載體中，位于兩個(gè)LTR的DNA片段和病毒基因組都會(huì)穩(wěn)定整合到靶細(xì)胞的基因組中。

通過改造優(yōu)化，我們的慢病毒載體刪除了與病毒包裝和轉(zhuǎn)導(dǎo)相關(guān)的基因（這些基因由輔助質(zhì)粒進(jìn)行表達(dá)，用于病毒包裝過程），使產(chǎn)生的慢病毒顆粒是復(fù)制缺陷型的。即包裝的病毒只具有轉(zhuǎn)導(dǎo)靶細(xì)胞的能力，而無法在靶細(xì)胞中進(jìn)行大量復(fù)制，因而具有很高的生物安全性。

關(guān)于慢病毒基因表達(dá)載體的更多信息，請(qǐng)參考以下文獻(xiàn)。

我們目前采用的是第三代慢病毒包裝載體系統(tǒng)。經(jīng)優(yōu)化，該載體在大腸桿菌體內(nèi)具有很高的拷貝數(shù)，包裝的活病毒具有很高的滴度，對(duì)大多數(shù)宿主細(xì)胞具有高效的轉(zhuǎn)導(dǎo)能力，能有效地把載體整合到靶細(xì)胞基因組并實(shí)現(xiàn)外源基因的高水平表達(dá)。

外源基因的穩(wěn)定整合：常規(guī)質(zhì)粒轉(zhuǎn)染只能實(shí)現(xiàn)外源基因的瞬時(shí)表達(dá)，這種外源基因會(huì)隨著宿主細(xì)胞的分裂而不斷丟失，在快速分裂的細(xì)胞中顯得尤為顯著。相反的是，慢病毒轉(zhuǎn)導(dǎo)的目的基因能穩(wěn)定地整合到宿主細(xì)胞的染色體中 ，因而會(huì)隨著宿主細(xì)胞的分裂而穩(wěn)定遺傳。

滴度高：我們的病毒載體可以包裝出高滴度的病毒。我們提供的病毒包裝服務(wù)，病毒滴度可以達(dá)到>10⁹ TU/ml。在這樣的病毒滴度下，如果選擇合適的劑量去轉(zhuǎn)導(dǎo)體外培養(yǎng)的哺乳動(dòng)物細(xì)胞，則轉(zhuǎn)導(dǎo)效率可接近100%。

宿主范圍廣泛：我們的病毒包裝系統(tǒng)包裝出來的病毒含有VSV-G包膜蛋白，此蛋白擁有非常廣泛的親和性，可以轉(zhuǎn)導(dǎo)幾乎所有的哺乳動(dòng)物細(xì)胞，包括分裂細(xì)胞，非分裂細(xì)胞，原代細(xì)胞，穩(wěn)定細(xì)胞系，干細(xì)胞，分化細(xì)胞，貼壁細(xì)胞和懸浮細(xì)胞等各類哺乳動(dòng)物細(xì)胞，甚至還可以轉(zhuǎn)導(dǎo)一些非哺乳動(dòng)物細(xì)胞。使用傳統(tǒng)的轉(zhuǎn)染方式轉(zhuǎn)導(dǎo)神經(jīng)元細(xì)胞是非常難的，但是采用我們慢病毒載體系統(tǒng)可以輕易的實(shí)現(xiàn)神經(jīng)元細(xì)胞的轉(zhuǎn)導(dǎo)。相對(duì)于在某些細(xì)胞中具有較低轉(zhuǎn)導(dǎo)效率的腺病毒和不能用于非分裂細(xì)胞的逆轉(zhuǎn)錄病毒而言，利用我們的慢病毒包裝系統(tǒng)包裝出來的病毒具有廣泛的親和性。

靈活使用啟動(dòng)子：我們的慢病毒載體已經(jīng)過優(yōu)化，其5' LTR的啟動(dòng)子已進(jìn)行了自失活。因此，客戶可以靈活使用啟動(dòng)子來驅(qū)動(dòng)目的基因的表達(dá)。這相對(duì)于只能依賴自身5' LTR啟動(dòng)子的MMLV載體來說是一個(gè)巨大的優(yōu)勢(shì)。

基因拷貝數(shù)相對(duì)均一：通常情況下，采用病毒轉(zhuǎn)導(dǎo)的方式可以比較均一的將外源基因轉(zhuǎn)入靶細(xì)胞中，而傳統(tǒng)的質(zhì)粒轉(zhuǎn)染則呈現(xiàn)出較高的不均一性，導(dǎo)致某些細(xì)胞會(huì)獲得較多拷貝質(zhì)粒而某些則會(huì)獲得較少甚至完全沒有。

體內(nèi)外實(shí)驗(yàn)均有效：我們的載體不僅擁有良好的體外細(xì)胞轉(zhuǎn)導(dǎo)能力，同樣適用于體內(nèi)活體動(dòng)物實(shí)驗(yàn)。

安全性：我們的病毒載體系統(tǒng)具備了以下兩大特點(diǎn)，因而具有非常高的安全性。一、病毒包裝和轉(zhuǎn)導(dǎo)所必需的基因由三個(gè)輔助質(zhì)粒分開表達(dá)。二、5' LTR的啟動(dòng)子自失活。因此，在進(jìn)行病毒包裝和病毒轉(zhuǎn)導(dǎo)的時(shí)候不會(huì)產(chǎn)生具有復(fù)制能力的病毒顆粒，使用我們的載體對(duì)人體的健康威脅也是最低的。

載體容量受限：野生型的慢病毒基因組大小約為9.2 kb，而在我們的慢病毒載體中，病毒包裝和轉(zhuǎn)導(dǎo)的必要元件約為2.8 kb，余下6.4 kb的空間容納客戶的目的序列。當(dāng)病毒載體超過以上大小限制，病毒的包裝滴度將會(huì)大大降低。我們的慢病毒載體除了可以插入靶基因的序列外，還可以插入啟動(dòng)子和篩選標(biāo)記等載體元件。如果目的基因和這些載體元件長(zhǎng)度超過了6.4 kb，病毒的產(chǎn)量有可能會(huì)明顯下降。

技術(shù)復(fù)雜：使用慢病毒載體時(shí)，需要在包裝細(xì)胞中產(chǎn)生活病毒，然后測(cè)定病毒滴度。因此慢病毒轉(zhuǎn)染相對(duì)于常規(guī)質(zhì)粒轉(zhuǎn)染，技術(shù)難度更高，周期更長(zhǎng)。

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.