用LMP1构建重组慢病毒载体及建立转基因小鼠模型
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摘要
EB病毒与多种人类肿瘤相关，尤其与鼻咽癌密切相关，但由于缺乏实验动物学方面的证据，EB病毒在鼻咽癌的发生、发展中所起的作用一直是研究的热点。但至今尚未能用EB病毒或其致瘤基因建立鼻咽癌动物模型，分析原因，我们认为主要与没有利用鼻咽组织相对特异性启动子有关。为了解决这一问题，本研究利用嗜上皮细胞特异性启动子ED-L2，携带增强型绿色荧光蛋白报告基因(enhanced green fluorescent protein,EGFP)与EB病毒潜伏膜蛋白(LMP1)，构建融合蛋白基因载体。本研究拟分别构建ED-L2-EGFP慢病毒表达载体、ED-L2-N-LMP1-EGFP慢病毒表达载体、ED-L2-B-LMP1-EGFP慢病毒表达载体；将EB病毒潜伏膜蛋白(LMP1)与增强型绿色荧光蛋白(EGFP)作为目的基因，通过显微注射技术，建立携带靶基因的转基因小鼠，为进一步建立鼻咽癌动物模型探索新途径。
研究内容及结果如下：
一、成功构建了ED-L2-EGFP慢病毒载体、ED-L2-N-LMP1-EGFP慢病毒载体、ED-L2-B-LMP1-EGFP慢病毒表达载体 以pED-L2-N-LMP1质粒为模板，通过PCR法扩增ED-L2启动子序列，上游5′端引入PacI酶切位点，下游5′端引入BamHI酶切位点，PCR产物胶回收纯化与pUC118载体16℃进行连接。利用基因重组技术，首先将ED-L2启动子序列克隆入慢病毒载体，然后分别将N-LMP1及B-LMP1克隆入含ED-L2启动子的慢病毒载体中。抽提质粒，重组载体酶切鉴定、基因测序，选
取正向插入克隆用于下一步研究。
二、重组载体体外转染实验重组载体分别转染5一8F、6一10B、CNEI、
293T细胞，在倒置荧光显微镜下观察，可见绿色荧光蛋白表达，说明载
体构建成功。提取转染细胞总RNA，通过RT一PcR检测，可见LMPI基
因表达。转染细胞免疫组织化学检测为阳性，证明LMPI基因在癌上皮
细胞水平有转录及蛋白质表达。而转染鼠成纤维细胞NIH3T3，未见目的
基因表达。提示ED一LZ启动子能够有效引导靶基因在上皮细胞的表达。
三、慢病毒载体转基因小鼠模型的建立利用ED一LZ一N一LMPI一EGFP
慢病毒载体、ED一LZ一B一LMPI一EGFP慢病毒载体，分别与么8.9及水泡性
口炎病毒糖蛋白(v esieular stomatitis virus glycoprotein，vsvo)共同转染
293T包装细胞，转染后60小时，收集上清，超速离心，浓缩病毒。用
适量PBS重悬病毒，利用显微注射法，将适量病毒悬液注射入小鼠受精
卵细胞，将注射后成活的受精卵细胞移入假孕雌鼠输卵管壶腹部，得到了
3只ED一LZ一N一LMPI一EGFP慢病毒转基因小鼠。
本研究主要创新点如下:
(一)首次将慢病毒载体、ED一LZ启动子、N一LMPI及B一LMPI有效
重组并成功构建了ED一LZ一EGFP、ED一LZ一N一LMPI一EGFP及ED一LZ-
B一LMPI一EGFP新型慢病毒载体;
(二)在细胞和分子水平对所构建的载体进行了验证并证明ED一LZ启
动子能有效引导靶基因在上皮细胞表达;
(三)利用带有鼻咽部特异启动子及致瘤靶基因的重组慢病毒载体系
统和显微注射技术，建立了慢病毒转基因小鼠模型并获得3只ED一LZ
一LMPI一EGFP慢病毒转基因小鼠。
本研究为建立鼻咽癌可视化动物模型奠定了良好的实验基础
Epstein-Barr virus (EBV) is associated with a number of human malignancies, especlialy with nasopharyngeal carcinoma (NPC). because of deficiency of animal model. However, due to lacking of evidence of animal models, the role of EB virus in NPC occurrence and development remains in researching hostspot .The reason for this may be that the regulators could not direct the transgene to be efficiently expressed in mouse nasopharynx. In view of this, the feasible strategy to get EBV -related transgenic model of NPC might be to select some appropriate regulators, which can lead target gene to be expressed efficiently and specifically in nasopharynx. In order to resolve this problem We took the following studies:
1. Expression and evaluation of recombinant LMP1 lentiviral vector. ED-L2 promoter was amplified by polymerase chain reaction (PCR) from pED-L2-N-LMPl vector template. PacI enzymed cite was inserted into 5 ' of forward primer, and BamHI enzymed cite was inserted into 5 downstream primer. Then production of PCR was ligated with pUC118 vector. Using the DNA recombinant techniques, two eukaryotic expression vectors N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were constructed respectively, both target genes N-LMP1 and B-LMP1 inserted in sense orientation were confirmed and by restriction endonuclease digestion analysis.
2. Gene transfection in vitro and analysis of transgenic vectors .the N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were transferred into CNE1, 5-8F, 6-10B and 293T cells. All of cell lines with N-LMP1 lentiviral
-4-
vector and B-LMP1 lentiviral vector transient transfection was investigated under fluorescence microscope, and the expression of enhanced green fluorescent protein could be observed. Reverse transcriptase PCR was used to detect the expression of LMP1 gene at the level of messenger RNA (mRNA) and so did immunohistochemical method to test the expression of LMP1 gene. Results showed the target gene could express correctly in host cells. These two identified vectors were also transferred into mouse fibroblasts NIH3T3 cells. The target gene couldn't express in NIH3T3 cells and blank control.
3. Establishment of transgenic mouse model. Using ED-L2-N-LMP1-EGFP lentiviral vector; 8.9; VSVG (vesicular stomatitis virus glycoprotein, VSVG) -pseudotyped were transferred together into 293T cells, the viral supernatant is ready for collection 60 hours after transfection. Adjust centrifuge to spin for 90 minutes at 4 25000 rpm, When spin is finished, remove all traces of supernatant, then add 100 l of cold PBS, keep tubes at 4 癈 for at least 12 hours to dissolve the pellet. The virus was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviduct of pseudo-pregnant female mice, thus the founder transgenic mice were obtained and their biological characteristic were observed by PCR and Southern blot analysis.
hi this study, we construct the vector using specific nasopharynx tissue promoter ED-L2 carrying the enhanced green fluorescent protein (EGFP) gene driven by a specific nasopharynx tissue expressing promoter, To construct ED-L2-EGFP lentiviral expression vector, ED-L2-N-LMP1-EGFP lentiviral expression vector and ED-L2-B-LMP1-EGFP lentiviral expression vector. Produce transgenic mouse model. The transgenic mice were built by microinjection. The DNA was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviducts of pseudo-pregnant recipient female mice, we obtained 3 founder mice of the ED-L2-N-LMP1-EGFP lentiviral vector group, and these mice were examined by PCR and Southern blot analysis. All of them were positive for gene integration.
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研究内容及结果如下：
一、成功构建了ED-L2-EGFP慢病毒载体、ED-L2-N-LMP1-EGFP慢病毒载体、ED-L2-B-LMP1-EGFP慢病毒表达载体 以pED-L2-N-LMP1质粒为模板，通过PCR法扩增ED-L2启动子序列，上游5′端引入PacI酶切位点，下游5′端引入BamHI酶切位点，PCR产物胶回收纯化与pUC118载体16℃进行连接。利用基因重组技术，首先将ED-L2启动子序列克隆入慢病毒载体，然后分别将N-LMP1及B-LMP1克隆入含ED-L2启动子的慢病毒载体中。抽提质粒，重组载体酶切鉴定、基因测序，选
取正向插入克隆用于下一步研究。
二、重组载体体外转染实验重组载体分别转染5一8F、6一10B、CNEI、
293T细胞，在倒置荧光显微镜下观察，可见绿色荧光蛋白表达，说明载
体构建成功。提取转染细胞总RNA，通过RT一PcR检测，可见LMPI基
因表达。转染细胞免疫组织化学检测为阳性，证明LMPI基因在癌上皮
细胞水平有转录及蛋白质表达。而转染鼠成纤维细胞NIH3T3，未见目的
基因表达。提示ED一LZ启动子能够有效引导靶基因在上皮细胞的表达。
三、慢病毒载体转基因小鼠模型的建立利用ED一LZ一N一LMPI一EGFP
慢病毒载体、ED一LZ一B一LMPI一EGFP慢病毒载体，分别与么8.9及水泡性
口炎病毒糖蛋白(v esieular stomatitis virus glycoprotein，vsvo)共同转染
293T包装细胞，转染后60小时，收集上清，超速离心，浓缩病毒。用
适量PBS重悬病毒，利用显微注射法，将适量病毒悬液注射入小鼠受精
卵细胞，将注射后成活的受精卵细胞移入假孕雌鼠输卵管壶腹部，得到了
3只ED一LZ一N一LMPI一EGFP慢病毒转基因小鼠。
本研究主要创新点如下:
(一)首次将慢病毒载体、ED一LZ启动子、N一LMPI及B一LMPI有效
重组并成功构建了ED一LZ一EGFP、ED一LZ一N一LMPI一EGFP及ED一LZ-
B一LMPI一EGFP新型慢病毒载体;
(二)在细胞和分子水平对所构建的载体进行了验证并证明ED一LZ启
动子能有效引导靶基因在上皮细胞表达;
(三)利用带有鼻咽部特异启动子及致瘤靶基因的重组慢病毒载体系
统和显微注射技术，建立了慢病毒转基因小鼠模型并获得3只ED一LZ
一LMPI一EGFP慢病毒转基因小鼠。
本研究为建立鼻咽癌可视化动物模型奠定了良好的实验基础
Epstein-Barr virus (EBV) is associated with a number of human malignancies, especlialy with nasopharyngeal carcinoma (NPC). because of deficiency of animal model. However, due to lacking of evidence of animal models, the role of EB virus in NPC occurrence and development remains in researching hostspot .The reason for this may be that the regulators could not direct the transgene to be efficiently expressed in mouse nasopharynx. In view of this, the feasible strategy to get EBV -related transgenic model of NPC might be to select some appropriate regulators, which can lead target gene to be expressed efficiently and specifically in nasopharynx. In order to resolve this problem We took the following studies:
1. Expression and evaluation of recombinant LMP1 lentiviral vector. ED-L2 promoter was amplified by polymerase chain reaction (PCR) from pED-L2-N-LMPl vector template. PacI enzymed cite was inserted into 5 ' of forward primer, and BamHI enzymed cite was inserted into 5 downstream primer. Then production of PCR was ligated with pUC118 vector. Using the DNA recombinant techniques, two eukaryotic expression vectors N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were constructed respectively, both target genes N-LMP1 and B-LMP1 inserted in sense orientation were confirmed and by restriction endonuclease digestion analysis.
2. Gene transfection in vitro and analysis of transgenic vectors .the N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were transferred into CNE1, 5-8F, 6-10B and 293T cells. All of cell lines with N-LMP1 lentiviral
-4-
vector and B-LMP1 lentiviral vector transient transfection was investigated under fluorescence microscope, and the expression of enhanced green fluorescent protein could be observed. Reverse transcriptase PCR was used to detect the expression of LMP1 gene at the level of messenger RNA (mRNA) and so did immunohistochemical method to test the expression of LMP1 gene. Results showed the target gene could express correctly in host cells. These two identified vectors were also transferred into mouse fibroblasts NIH3T3 cells. The target gene couldn't express in NIH3T3 cells and blank control.
3. Establishment of transgenic mouse model. Using ED-L2-N-LMP1-EGFP lentiviral vector; 8.9; VSVG (vesicular stomatitis virus glycoprotein, VSVG) -pseudotyped were transferred together into 293T cells, the viral supernatant is ready for collection 60 hours after transfection. Adjust centrifuge to spin for 90 minutes at 4 25000 rpm, When spin is finished, remove all traces of supernatant, then add 100 l of cold PBS, keep tubes at 4 癈 for at least 12 hours to dissolve the pellet. The virus was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviduct of pseudo-pregnant female mice, thus the founder transgenic mice were obtained and their biological characteristic were observed by PCR and Southern blot analysis.
hi this study, we construct the vector using specific nasopharynx tissue promoter ED-L2 carrying the enhanced green fluorescent protein (EGFP) gene driven by a specific nasopharynx tissue expressing promoter, To construct ED-L2-EGFP lentiviral expression vector, ED-L2-N-LMP1-EGFP lentiviral expression vector and ED-L2-B-LMP1-EGFP lentiviral expression vector. Produce transgenic mouse model. The transgenic mice were built by microinjection. The DNA was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviducts of pseudo-pregnant recipient female mice, we obtained 3 founder mice of the ED-L2-N-LMP1-EGFP lentiviral vector group, and these mice were examined by PCR and Southern blot analysis. All of them were positive for gene integration.
-5-
引文
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