以GFP为报告基因的多基因共表达慢病毒载体系统的构建

发布时间：2018-05-07 00:03

本文选题：基因治疗 + 慢病毒载体　；参考：《郑州大学》2009年硕士论文

【摘要】：神经细胞的转基因技术是不仅是研究神经系统疾病的分子病理机制的重要手段,而且为神经系统疾病的基因治疗带来了新的希望。其关键之一就是选择良好的转基因载体。近年来慢病毒载体以其独特的优势而越来越被人们所关注。慢病毒载体(LV,lentiviral vector)来源于人类免疫缺陷病毒1型(HIV-1),其致病基因已经被剔除,它能感染包括神经元在内的非分裂细胞、感染效率高、免疫原性低,可携带转基因整合到宿主细胞基因组内而长期稳定地表达所携带目的基因。因大多数神经系统疾病是由多个基因共同作用所致,故进行基因治疗时需要选择能够同时上调(强启动子驱动)目的基因或下调(siRNA基因沉默)多个靶基因的转基因载体。因此当前慢病毒载体研究的一个方向即是构建多基因共表达载体。此外,因为慢病毒感染宿主细胞时无明显的感染标记,故进行滴度测定时极为不便,目前尚缺快速稳定的滴度测定方法。针对上述问题,本研究利用分子克隆方法构建了一套多基因共表达慢病毒载体构建系统,此系统可用以构建多siRNA、报告基因GFP和目的基因共表达的慢病毒载体。可将GFP作为感染标记,采用标准的TCID_(50)法计算病毒滴度,建立了一种方便快捷的慢病毒滴度测定方法,为神经系统疾病的研究和治疗建立了一个实用工具。目的: 1.构建多个siRNA表达盒,GFP及目的基因共表达的慢病毒载体构建系统pLKO-M和pSi-shutle。 2.和慢病毒包膜质粒与辅助质粒共转染293T细胞产生表达GFP的慢病毒颗粒并建立慢病毒滴度的TCID_(50)快速测定方法。方法: 1.现有慢病毒载体pLKO-1-puro的多克隆位点的改建:设计并合成新的多克隆位点(AgeI、EcoRI、XbaI、SalI和MluI)序列,退火后连接到经AgeI和EcoRI双酶切的pLKO-1-puro载体质粒上,形成重组质粒pLKO-1-puro-MCS;转化大肠杆菌(escherichia coli,E.coli)DH5α感受态细胞,筛选阳性菌落、扩增后提取质粒,MluI和BamHI双酶切鉴定。 2.CMV启动子-GFP-IRES-MCS元件克隆至pLKO-1-puro-MCS:以pGFP-IRES为模板设计引物,用高保真DNA聚合酶扩增出约2.4kb片段,XbaI和SalI酶切后克隆至pLKO-1-puro-MCS的XbaI和SalI位点处,XbaI和lSalI酶切鉴定。 3.配套质粒pSi-shutle的构建:设计INPTEN1和INPTEN2引物,PCR扩增pSilencer2.0,得到421bp片段,将其克隆至pENTR-U6-con,得pSi-shutle,SalI和XbaI酶切鉴定阳性菌落。 4.用脂质体2000将重组载体质粒PLKO-M、辅助质粒pCMV-dR8.2 dvpr和包膜质粒pCMV-VSV-G共同转染293T细胞,产生具有感染能力的慢病毒颗粒,在25000rpm、4℃条件下离心90min浓缩病毒颗粒。 5.TCID_(50)法测定慢病毒滴度:将病毒颗粒进行系列稀释后感染2个96孔板内Vero细胞,48小时后在倒置荧光显微镜下计数产生绿色荧光的孔数并计算滴度。结果: 1.含新的多克隆位点(MCS)的寡核苷酸链被成功克隆到慢病毒载体质粒pLKO-1-puro中,得pLKO-1-puro-MCS,经过酶切鉴定证明构建成功。 2.CMV启动子-GFP-IRES-MCS元件被成功克隆至pLKO-1-puro-MCS,酶切鉴定正确。 3.pSilencer2.0质粒上的siRNA表达盒被成功克隆至pENTR-u6-con,酶切和测序鉴定正确。 4.三质粒共转染293T 48小时后可见GFP表达,72小时后收获慢病毒颗粒并进行了浓缩。 5.将浓缩后的病毒系列稀释后感染2个96孔板内Vero细胞,48小时后出现绿色荧光,用TCID_(50)法计算得出浓缩后的病毒感染单位(IU)均值为6.47X10~6TU/ml。结论: 1.成功构建了siRNA表达盒和GFP-目的基因共表达的慢病毒载体pLKO-M。 2.成功构建了pLKO-M的配套质粒psi-shuttle,在其辅助下可以构建多siRNA、GFP和目的基因共表达的慢病毒载体 3.建立了以绿色荧光作为感染标记,用TCID_(50)法测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 .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

【共引文献】