稳定表达siat7e基因的MDCK细胞构建

发布时间：2018-06-07 23:25

本文选题：FRT-LacZ基因 + 慢病毒载体　；参考：《西北民族大学》2016年硕士论文

【摘要】：犬肾细胞(Madin Darby canine kidney, MDCK)可替代鸡胚用于流感疫苗生产时病毒增殖,其悬浮培养能极大促进流感疫苗的规模化生产。siat7e基因的表达能有效的降低细胞的贴壁依赖性,便于细胞悬浮驯化。本研究通过慢病毒载体系统构建了含有反转酶识别位点稳定表达FRT-LacZ基因的MDCK细胞(Flp-In MDCK)。然后共转染pcDNA5/FRT-siat7e载体与Flp重组酶表达载体pOG44,筛选获得了稳定表达siat7e基因的重组细胞MDCK-siat7e。并对其进行了悬浮驯化,获得了以下研究结果：1.构建了稳定表达FRT-LacZ基因的Flp-In MDCK细胞构建了含有3138 bp FRT-LacZ基因的慢病毒重组表达质粒pLVX-FRT-LacZ-PGK-Puro。与包装质粒(pLP1, pLP2, pLP/VSVG)共转染293FT细胞获得重组慢病毒rLV-FRT-LacZ,病毒滴度达4×107TU/mL。重组慢病毒感染MDCK细胞,经嘌呤霉素筛选2周后,β-半乳糖苷酶原位染色呈蓝色,表明获得了稳定表达FRT-LacZ基因的Flp-In MDCK细胞。2.构建了可定点整合siat7e基因表达载体pcDNA5.0/FRT-siat7e以EX-V1581-M03质粒为模板扩增出约1028 bp siat7e基因,双酶切后克隆至真核表达载体pcDNA5.0/FRT,构建了重组表达载体pcDNA5/FRT-siat7e。3.构建了七株稳定表达siat7e基因的MDCK-siat7e单克隆细胞使用脂质体将重组质粒pcDNA5/FRT-siat7e与重组酶表达质粒pOG44 以 1:9质量比共转染Flp-In MDCK细胞。通过Hygromycin B筛选获得表达siat7e基因MDCK-siat7e细胞。经β-半乳糖苷酶原位染色检测和siat7e基因mRNA的RT-PCR鉴定。采用有限稀释法进行单细胞克隆获得七株单克隆细胞系,命名为MDCK-siat7e (A、B、C、D、E、F、G)。实时荧光定量PCR检测表明,七株细胞中siat7e基因都得到稳定表达,其中MDCK-siat7e(G)细胞siat7e基因表达显著高于MDCK-siat7e(B、C、D、E) (p0.05),而MDCK-siat7e(G)与MDCK-siat7e(F、A)细胞中siat7e基因表达差异不显著(p0.05)。4. MDCK-siat7e细胞的悬浮驯化使用150 mL摇瓶以DMEM、SFM、DM/F12三种培养基分别添加1%NBS对MDCK-siat7e(G)细胞进行了悬浮驯化,其中血清浓度为1%的SFM培养基悬浮驯化效果较好。同时,siat7e基因的表达能显著减低MDCK细胞悬浮驯化过程中的结团现象。
[Abstract]:Madin Darby canine Kidney (MDCK) can be used to replace chicken embryo for virus proliferation in influenza vaccine production. The suspension culture can greatly promote the production of influenza vaccine on a large scale and the expression of .siat7e gene can effectively reduce cell adhesion. It is convenient for cell suspension and acclimation. In this study, a novel Lentivirus vector system was used to construct Flp-In MDCK cells containing reverse transcriptase recognition sites and stable expression of FRT-LacZ gene in MDCK cells. Then co-transfected pcDNA5 / FRT-siat7e vector and flp recombinant enzyme expression vector pOG44. the recombinant cell line MDCK-siat7eexpressing siat7e gene stably was obtained. The following results were obtained: 1: 1. Flp-In MDCK cells stably expressing FRT-LacZ gene were constructed, and lentivirus recombinant plasmid pLVX-FRT-LacZ-PGK-Puro. containing 3138 BP FRT-LacZ gene was constructed. The recombinant lentivirus rLV-FRT-LacZ was obtained by co-transfection with pLP1, pLP2and pLP / VSVG into 293FT cells. The titer of rLV-FRT-LacZ was 4 脳 107 TU / mL. After 2 weeks of purine mycin screening, 尾 -galactosidase was stained blue in situ, indicating that Flp-In MDCK cells expressing FRT-LacZ gene stably were obtained. A site-specific integrated siat7e gene expression vector pcDNA5.0 / FRT-siat7e was constructed. About 1028 BP siat7e gene was amplified from EX-V1581-M03 plasmid and cloned into eukaryotic expression vector pcDNA5.0 / FRT-siat7e.3.The recombinant expression vector pcDNA5 / FRT-siat7e.3was constructed and cloned into eukaryotic expression vector pcDNA5.0 / FRT-siat7e.3. Seven MDCK-siat7e monoclonal cells stably expressing siat7e gene were constructed. The recombinant plasmid pcDNA5 / FRT-siat7e and recombinant enzyme expression plasmid pOG44 were co-transfected into Flp-In MDCK cells by 1:9 mass ratio using liposome. MDCK-siat7e cells expressing siat7e gene were obtained by Hygromycin B screening. 尾-galactosidase in situ staining and RT-PCR identification of siat7e gene. Seven monoclonal cell lines named MDCK-siat7e (ADCK-siat7e) were obtained by single cell cloning with limited dilution method, and they were named MDCK-siat7e (ADCK-siat7e). The expression of siat7e gene in MDCK-siat7eG) cells was significantly higher than that in MDCK-siat7e7eG cells, while the expression of siat7e gene was not significantly different between MDCK-siat7eG) cells and MDCK-siat7eFA cells (p0.05n.4.The expression of siat7e gene in MDCK-siat7e7eG) cells was higher than that in MDCK-siat7e7eFAs cells (P < 0.05), but there was no significant difference in siat7e gene expression between MDCK-siat7eG cells and MDCK-siat7eFAs cells. The suspension acclimation of MDCK-siat7e cells was carried out by using 150 mL shaking flask and adding 1 NBS to DMEMS-SFMN / DMF12 medium respectively. The suspension acclimation of MDCK-siat7e7e cells was carried out on SFM medium with serum concentration of 1%. The suspension acclimation effect of SFM medium with 1% serum concentration was better. At the same time, the expression of siat7e gene can significantly reduce the formation of MDCK cells during suspension acclimation.
【学位授予单位】：西北民族大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

【相似文献】