發(fā)布時(shí)間：2018-05-07 00:03

  本文選題：基因治療 + 慢病毒載體　； 參考：《鄭州大學(xué)》2009年碩士論文

【摘要】：神經(jīng)細(xì)胞的轉(zhuǎn)基因技術(shù)是不僅是研究神經(jīng)系統(tǒng)疾病的分子病理機(jī)制的重要手段,而且為神經(jīng)系統(tǒng)疾病的基因治療帶來(lái)了新的希望。其關(guān)鍵之一就是選擇良好的轉(zhuǎn)基因載體。近年來(lái)慢病毒載體以其獨(dú)特的優(yōu)勢(shì)而越來(lái)越被人們所關(guān)注。慢病毒載體(LV,lentiviral vector)來(lái)源于人類免疫缺陷病毒1型(HIV-1),其致病基因已經(jīng)被剔除,它能感染包括神經(jīng)元在內(nèi)的非分裂細(xì)胞、感染效率高、免疫原性低,可攜帶轉(zhuǎn)基因整合到宿主細(xì)胞基因組內(nèi)而長(zhǎng)期穩(wěn)定地表達(dá)所攜帶目的基因。因大多數(shù)神經(jīng)系統(tǒng)疾病是由多個(gè)基因共同作用所致,故進(jìn)行基因治療時(shí)需要選擇能夠同時(shí)上調(diào)(強(qiáng)啟動(dòng)子驅(qū)動(dòng))目的基因或下調(diào)(siRNA基因沉默)多個(gè)靶基因的轉(zhuǎn)基因載體。因此當(dāng)前慢病毒載體研究的一個(gè)方向即是構(gòu)建多基因共表達(dá)載體。此外,因?yàn)槁《靖腥舅拗骷?xì)胞時(shí)無(wú)明顯的感染標(biāo)記,故進(jìn)行滴度測(cè)定時(shí)極為不便,目前尚缺快速穩(wěn)定的滴度測(cè)定方法。針對(duì)上述問題,本研究利用分子克隆方法構(gòu)建了一套多基因共表達(dá)慢病毒載體構(gòu)建系統(tǒng),此系統(tǒng)可用以構(gòu)建多siRNA、報(bào)告基因GFP和目的基因共表達(dá)的慢病毒載體�？蓪FP作為感染標(biāo)記,采用標(biāo)準(zhǔn)的TCID_(50)法計(jì)算病毒滴度,建立了一種方便快捷的慢病毒滴度測(cè)定方法,為神經(jīng)系統(tǒng)疾病的研究和治療建立了一個(gè)實(shí)用工具。 目的: 1.構(gòu)建多個(gè)siRNA表達(dá)盒,GFP及目的基因共表達(dá)的慢病毒載體構(gòu)建系統(tǒng)pLKO-M和pSi-shutle。 2.和慢病毒包膜質(zhì)粒與輔助質(zhì)粒共轉(zhuǎn)染293T細(xì)胞產(chǎn)生表達(dá)GFP的慢病毒顆粒并建立慢病毒滴度的TCID_(50)快速測(cè)定方法。 方法: 1.現(xiàn)有慢病毒載體pLKO-1-puro的多克隆位點(diǎn)的改建:設(shè)計(jì)并合成新的多克隆位點(diǎn)(AgeI、EcoRI、XbaI、SalI和MluI)序列,退火后連接到經(jīng)AgeI和EcoRI雙酶切的pLKO-1-puro載體質(zhì)粒上,形成重組質(zhì)粒pLKO-1-puro-MCS;轉(zhuǎn)化大腸桿菌(escherichia coli,E.coli)DH5α感受態(tài)細(xì)胞,篩選陽(yáng)性菌落、擴(kuò)增后提取質(zhì)粒,MluI和BamHI雙酶切鑒定。 2.CMV啟動(dòng)子-GFP-IRES-MCS元件克隆至pLKO-1-puro-MCS:以pGFP-IRES為模板設(shè)計(jì)引物,用高保真DNA聚合酶擴(kuò)增出約2.4kb片段,XbaI和SalI酶切后克隆至pLKO-1-puro-MCS的XbaI和SalI位點(diǎn)處,XbaI和lSalI酶切鑒定。 3.配套質(zhì)粒pSi-shutle的構(gòu)建:設(shè)計(jì)INPTEN1和INPTEN2引物,PCR擴(kuò)增pSilencer2.0,得到421bp片段,將其克隆至pENTR-U6-con,得pSi-shutle,SalI和XbaI酶切鑒定陽(yáng)性菌落。 4.用脂質(zhì)體2000將重組載體質(zhì)粒PLKO-M、輔助質(zhì)粒pCMV-dR8.2 dvpr和包膜質(zhì)粒pCMV-VSV-G共同轉(zhuǎn)染293T細(xì)胞,產(chǎn)生具有感染能力的慢病毒顆粒,在25000rpm、4℃條件下離心90min濃縮病毒顆粒。 5.TCID_(50)法測(cè)定慢病毒滴度:將病毒顆粒進(jìn)行系列稀釋后感染2個(gè)96孔板內(nèi)Vero細(xì)胞,48小時(shí)后在倒置熒光顯微鏡下計(jì)數(shù)產(chǎn)生綠色熒光的孔數(shù)并計(jì)算滴度。 結(jié)果: 1.含新的多克隆位點(diǎn)(MCS)的寡核苷酸鏈被成功克隆到慢病毒載體質(zhì)粒pLKO-1-puro中,得pLKO-1-puro-MCS,經(jīng)過酶切鑒定證明構(gòu)建成功。 2.CMV啟動(dòng)子-GFP-IRES-MCS元件被成功克隆至pLKO-1-puro-MCS,酶切鑒定正確。 3.pSilencer2.0質(zhì)粒上的siRNA表達(dá)盒被成功克隆至pENTR-u6-con,酶切和測(cè)序鑒定正確。 4.三質(zhì)粒共轉(zhuǎn)染293T 48小時(shí)后可見GFP表達(dá),72小時(shí)后收獲慢病毒顆粒并進(jìn)行了濃縮。 5.將濃縮后的病毒系列稀釋后感染2個(gè)96孔板內(nèi)Vero細(xì)胞,48小時(shí)后出現(xiàn)綠色熒光,用TCID_(50)法計(jì)算得出濃縮后的病毒感染單位(IU)均值為6.47X10~6TU/ml。 結(jié)論: 1.成功構(gòu)建了siRNA表達(dá)盒和GFP-目的基因共表達(dá)的慢病毒載體pLKO-M。 2.成功構(gòu)建了pLKO-M的配套質(zhì)粒psi-shuttle,在其輔助下可以構(gòu)建多siRNA、GFP和目的基因共表達(dá)的慢病毒載體 3.建立了以綠色熒光作為感染標(biāo)記,用TCID_(50)法測(cè)定慢病毒滴度的方法。
[Abstract]:The present study uses molecular cloning method to construct a multi - gene co - expression vector , which can infect non - dividing cells including neurons . It can infect non - dividing cells including neurons . It can infect non - dividing cells including neurons . It can be used to construct a multi - gene co - expression vector .

Purpose :

1 . constructing a plurality of siRNA expression cassettes , a lentivirus vector constructing system pLKO - M and a pSi - shutle which are co - expressed by GFP and a target gene .

2 . A rapid determination method of TCID _ ( 50 ) expressing GFP by co - transfection of two and two lentivirus envelope plasmids with helper plasmids to produce lentivirus particles expressing GFP and establishing a slow virus titer .

Method :

1 . Reconstruction of the multiple cloning site of the existing slow virus vector pLKO - 1 - puro : designing and synthesizing the new multi - cloning site ( AIGI , EcoRI , Xba , Sal I and MluI ) , annealing , and connecting to the pLKO - 1 - puro vector plasmid digested with the two enzymes of the I and EcoRI to form the recombinant plasmid pLKO - 1 - puro - MCS ; transforming Escherichia coli ( E . coli ) DH5.alpha competent cells , screening the positive bacteria , and extracting the plasmids , MluI and BamHI .

2 . Cloning of the CMV promoter - GFP - based - MCS element to pLKO - 1 - puro - MCS : primer was designed using pGFP as a template , and about 2.4 kb fragment was amplified with a high - fidelity DNA polymerase , and digested with a high - fidelity DNA polymerase , and cloned into a pLKO - 1 - puro - MCS site at the Xba and Sal I sites , and digested with Xba and lSal .

3 . Construction of the matching plasmid pSi - shutle : designing INPTEN1 and INPTEN2 primers , amplifying pSilencer2.0 by PCR , and cloning to pENTR - 6 - con to obtain pSi - shutle , Sal I and Xba enzyme .

4 . The recombinant vector plasmid PLKO - M , the helper plasmid pCMV - dR8 . 2 dvpr and the envelope plasmid pCMV - vsv - G were co - transfected with liposome 2000 to produce lentivirus particles with the ability of infection , and the virus particles were concentrated at 25000rpm and 4.degree . C.for 90 min .

5 . Test the titer of lentivirus by TCID _ ( 50 ) . After series dilution of the virus particles , the Vero cells in two 96 - well plates were infected . After 48 hours , the number of green fluorescence was counted and the titer was calculated under the inverted fluorescence microscope .

Results :

1 . An oligonucleotide chain containing a new multiple cloning site ( MCS ) was successfully cloned into the lentivirus vector plasmid pLKO - 1 - puro to obtain pLKO - 1 - puro - MCS , which proved successful in construction .

2 . The CMV promoter - GFP - EGFP - MCS element was successfully cloned into pLKO - 1 - puro - MCS , and the enzyme digestion was correct .

3 . siRNA expression cassette on pSilencer2.0 plasmid was successfully cloned into pENTR - u6 - con .

4 . GFP expression was observed after co - transfection of three plasmids for 48 hours . After 72 hours , the lentivirus particles were harvested and concentrated .

5 . After diluting the concentrated virus series , Vero cells in two 96 - well plates were infected , green fluorescence appeared after 48 hours , and the mean value of the infected unit ( IU ) after concentration was 6.47X10 - 6TU / ml by TCID _ ( 50 ) method .

Conclusion :

1 . The lentivirus vector pLKO - M co - expressed by siRNA expression cassette and GFP - target gene was successfully constructed .

2 . pLKO - M is successfully constructed with pLKO - M plasmid ppsi - shuttle , which can construct a slow virus vector co - expressed by siRNA , GFP and target gene under the aid of the pLKO - M .

3 . A method for the determination of slow virus titer by TCID _ ( 50 ) method was established .

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郭國(guó)英;謝國(guó)良;何國(guó)清;孔彥平;王毅剛;;HSV-tk聯(lián)合GCV自殺系統(tǒng)對(duì)肝癌細(xì)胞株Huh-7殺傷作用的研究[J];浙江理工大學(xué)學(xué)報(bào);2011年02期

相關(guān)會(huì)議論文 前1條

1 樊寶良;;特異性識(shí)別雞卵清蛋白基因3'非編碼區(qū)序列的鋅指核酸酶基因的設(shè)計(jì)裝配及初步功能驗(yàn)證[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)動(dòng)物繁殖學(xué)分會(huì)第十五屆學(xué)術(shù)研討會(huì)論文集（上冊(cè)）[C];2010年

相關(guān)博士學(xué)位論文 前4條

1 張國(guó)壽;酵母雙雜交篩選與TCTP相互作用的蛋白及其功能的研究[D];福建醫(yī)科大學(xué);2011年

2 王琪影;靶向RhoC基因慢病毒載體構(gòu)建及對(duì)黑色素瘤細(xì)胞生物學(xué)行為和DTIC耐藥性影響[D];鄭州大學(xué);2011年

3 曹明媚;庚型肝炎病毒作為復(fù)制型基因治療載體及小干擾RNA對(duì)病毒表達(dá)復(fù)制抑制作用的研究[D];第二軍醫(yī)大學(xué);2004年

4 張強(qiáng);表達(dá)亞洲1型口蹄疫病毒P1-2A3C基因的山羊痘病毒弱毒株構(gòu)建及其復(fù)制非必需區(qū)篩選[D];中國(guó)農(nóng)業(yè)科學(xué)院;2007年

相關(guān)碩士學(xué)位論文 前6條

1 馮R，

本文編號(hào)：1854484