本文選題：FRT-LacZ基因 + 慢病毒載體　； 參考：《西北民族大學(xué)》2016年碩士論文

【摘要】：犬腎細(xì)胞(Madin Darby canine kidney, MDCK)可替代雞胚用于流感疫苗生產(chǎn)時(shí)病毒增殖,其懸浮培養(yǎng)能極大促進(jìn)流感疫苗的規(guī)�；a(chǎn)。siat7e基因的表達(dá)能有效的降低細(xì)胞的貼壁依賴性,便于細(xì)胞懸浮馴化。本研究通過慢病毒載體系統(tǒng)構(gòu)建了含有反轉(zhuǎn)酶識別位點(diǎn)穩(wěn)定表達(dá)FRT-LacZ基因的MDCK細(xì)胞(Flp-In MDCK)。然后共轉(zhuǎn)染pcDNA5/FRT-siat7e載體與Flp重組酶表達(dá)載體pOG44,篩選獲得了穩(wěn)定表達(dá)siat7e基因的重組細(xì)胞MDCK-siat7e。并對其進(jìn)行了懸浮馴化,獲得了以下研究結(jié)果：1.構(gòu)建了穩(wěn)定表達(dá)FRT-LacZ基因的Flp-In MDCK細(xì)胞構(gòu)建了含有3138 bp FRT-LacZ基因的慢病毒重組表達(dá)質(zhì)粒pLVX-FRT-LacZ-PGK-Puro。與包裝質(zhì)粒(pLP1, pLP2, pLP/VSVG)共轉(zhuǎn)染293FT細(xì)胞獲得重組慢病毒rLV-FRT-LacZ,病毒滴度達(dá)4×107TU/mL。重組慢病毒感染MDCK細(xì)胞,經(jīng)嘌呤霉素篩選2周后,β-半乳糖苷酶原位染色呈藍(lán)色,表明獲得了穩(wěn)定表達(dá)FRT-LacZ基因的Flp-In MDCK細(xì)胞。2.構(gòu)建了可定點(diǎn)整合siat7e基因表達(dá)載體pcDNA5.0/FRT-siat7e以EX-V1581-M03質(zhì)粒為模板擴(kuò)增出約1028 bp siat7e基因,雙酶切后克隆至真核表達(dá)載體pcDNA5.0/FRT,構(gòu)建了重組表達(dá)載體pcDNA5/FRT-siat7e。3.構(gòu)建了七株穩(wěn)定表達(dá)siat7e基因的MDCK-siat7e單克隆細(xì)胞使用脂質(zhì)體將重組質(zhì)粒pcDNA5/FRT-siat7e與重組酶表達(dá)質(zhì)粒pOG44 以 1:9質(zhì)量比共轉(zhuǎn)染Flp-In MDCK細(xì)胞。通過Hygromycin B篩選獲得表達(dá)siat7e基因MDCK-siat7e細(xì)胞。經(jīng)β-半乳糖苷酶原位染色檢測和siat7e基因mRNA的RT-PCR鑒定。采用有限稀釋法進(jìn)行單細(xì)胞克隆獲得七株單克隆細(xì)胞系,命名為MDCK-siat7e (A、B、C、D、E、F、G)。實(shí)時(shí)熒光定量PCR檢測表明,七株細(xì)胞中siat7e基因都得到穩(wěn)定表達(dá),其中MDCK-siat7e(G)細(xì)胞siat7e基因表達(dá)顯著高于MDCK-siat7e(B、C、D、E) (p0.05),而MDCK-siat7e(G)與MDCK-siat7e(F、A)細(xì)胞中siat7e基因表達(dá)差異不顯著(p0.05)。4. MDCK-siat7e細(xì)胞的懸浮馴化使用150 mL搖瓶以DMEM、SFM、DM/F12三種培養(yǎng)基分別添加1%NBS對MDCK-siat7e(G)細(xì)胞進(jìn)行了懸浮馴化,其中血清濃度為1%的SFM培養(yǎng)基懸浮馴化效果較好。同時(shí),siat7e基因的表達(dá)能顯著減低MDCK細(xì)胞懸浮馴化過程中的結(jié)團(tuán)現(xiàn)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.
【學(xué)位授予單位】：西北民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78

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本文編號：1993269