本文選題：桿狀病毒 + 流式細(xì)胞儀　； 參考：《武漢工程大學(xué)》2012年碩士論文

【摘要】：桿狀病毒表達(dá)載體系統(tǒng)作為最具發(fā)展前景和應(yīng)用價(jià)值的表達(dá)載體系統(tǒng)之一，被廣泛用于重組蛋白的生產(chǎn)、疫苗的研制以及制備生物殺蟲劑。但由于桿狀病毒感染過程十分復(fù)雜，現(xiàn)階段又沒有一個(gè)準(zhǔn)確的、有效的感染動(dòng)力學(xué)數(shù)學(xué)模型來模擬這一過程，從而給工業(yè)化生產(chǎn)帶來了諸多不便。建立一個(gè)準(zhǔn)確的病毒感染動(dòng)力學(xué)模型，需要精確測定這一過程中的各項(xiàng)參數(shù)。而細(xì)胞周期與細(xì)胞生長時(shí)間對(duì)病毒復(fù)制能力也有著非常重要的影響。建立準(zhǔn)確的病毒感染動(dòng)力學(xué)模型，必需把這兩個(gè)因素加入到模型中，因此本論文也對(duì)這兩個(gè)因素對(duì)病毒復(fù)制能力的影響做了較為深入的研究。 本論文用SYBR Green Ⅰ熒光染料對(duì)自由病毒DNA進(jìn)行染色；用抗GP64蛋白的單克隆抗體（AcV1）結(jié)合自由病毒粒子，然后用熒光二抗標(biāo)記AcV1從而間接標(biāo)記感染細(xì)胞。這樣被標(biāo)記上熒光的病毒與細(xì)胞就能用流式細(xì)胞儀精確測定病毒滴度和感染細(xì)胞百分率。最終得到如下結(jié)論：（1）通過對(duì)終點(diǎn)稀釋法和流式檢測法的比較，得到大約5個(gè)BV病毒粒子等于1個(gè)TCID_(50)感染單位。（2）通過對(duì)細(xì)胞吸附病毒過程中病毒滴度的減少量和細(xì)胞的總量之間的關(guān)系得出：一個(gè)Sf9細(xì)胞的飽和病毒吸附量為50個(gè)BV，那么當(dāng)MOI值大于50BVs/cell時(shí)就為高感染。（3）通過對(duì)病毒滴度的變化關(guān)系得出：細(xì)胞吸附病毒的過程要持續(xù)6h；在感染后3h，，細(xì)胞胞吞病毒的速率開始大于細(xì)胞吸附病毒的速率。（4）通過對(duì)“陰性最高熒光強(qiáng)度分界法”、“陰性曲線與陽性曲線交叉法”和“匹配面積相減法”，這三種分析方法的比較，得出在這三種分析方法中用“匹配面積相減法”來分析感染細(xì)胞的百分率是最為準(zhǔn)確的。 本論文對(duì)生長時(shí)間、細(xì)胞周期對(duì)細(xì)胞活性和病毒感染能力的影響也進(jìn)行了研究。生長時(shí)間對(duì)細(xì)胞活性和病毒感染能力的影響是通過采集延滯期、對(duì)數(shù)生長期和穩(wěn)定期的細(xì)胞，用MTT法研究三個(gè)不同時(shí)期的細(xì)胞活性的強(qiáng)弱關(guān)系，并用病毒感染，來確定三個(gè)不同時(shí)期的細(xì)胞被感染后的病毒復(fù)制能力的強(qiáng)弱。最終得到如下結(jié)論：（1）用MTT法研究延滯期、對(duì)數(shù)生長期和穩(wěn)定期這三個(gè)不同時(shí)期的細(xì)胞活性強(qiáng)弱關(guān)系，得到細(xì)胞活性的關(guān)系是：對(duì)數(shù)生長期＞延滯期＞穩(wěn)定期。（2）延滯期、對(duì)數(shù)生長期和穩(wěn)定期這三個(gè)不同時(shí)期的細(xì)胞被感染后的病毒復(fù)制能力的強(qiáng)弱關(guān)系為為：對(duì)數(shù)生長期＞延滯期＞穩(wěn)定期 細(xì)胞周期對(duì)細(xì)胞活性和病毒感染能力的影響，首先是用諾考達(dá)唑?qū)?xì)胞進(jìn)行同步化，然后采集初始細(xì)胞周期分布不同的細(xì)胞（G1期比例最大的細(xì)胞、S期比例最大的細(xì)胞和G_2/M期比例最大的細(xì)胞）。實(shí)驗(yàn)用MTT法確定三個(gè)期的細(xì)胞活性的強(qiáng)弱關(guān)系為：G_1期＞S期＞G_2/M期，得到三個(gè)不同期的細(xì)胞被感染后的病毒復(fù)制能力的強(qiáng)弱關(guān)系為：G_1期＞S期＞G_2/M期。最后病毒感染對(duì)細(xì)胞周期也有著顯著的影響，當(dāng)Sf9細(xì)胞被AcMNPV病毒感染后，細(xì)胞周期會(huì)最終阻斷并累積在G_2/M期。
[Abstract]:Baculovirus expression vector system as one of the most promising and valuable carrier system, is widely used for recombinant protein production, vaccine development and preparation of bio pesticides. But because baculovirus infection process is very complicated, at present there is not an accurate, effective infection dynamics mathematical model to simulate the the process, which brings a lot of inconvenience to industrial production. To establish an accurate dynamic model of virus infection, need to accurately determine the parameters of this process. The cell cycle and cell growth time of virus replication ability is very important. To establish accurate dynamics model of virus infection, it is necessary to two the factors added to the model, so this paper also on the two factors which influence the replication ability of the virus to do a more in-depth study.
This paper used SYBR Green 1 fluorescent dye free virus DNA staining; using monoclonal antibody against GP64 protein (AcV1) combined with free virus particles, and then use the two fluorescent antibody labeling AcV1 indirectly labeled infected cells. This can be labeled virus and cell fluorescence on the precise determination of virus titer and infection of cells by flow cytometry cell instrument. Finally get the following conclusions: (1) comparing the end point dilution method and flow cytometry, about 5 BV of virus particles is equal to 1 TCID_ (50). (2) infection by virus on the cell adsorption process of virus titer and reduce the relationship between total amount of cells that a saturated virus adsorption of Sf9 cells was 50 BV, so when the MOI value is greater than 50BVs/cell for high infection. (3) through the relationship between the change of virus titer was obtained: the process of cell adsorption of virus to continue 6H After infection; 3h, the rate of cell endocytosis of virus was larger than cell adsorption of virus. (4) the highest fluorescence intensity of negative boundary method "," negative and positive curve curve intersection method "and" matching area subtraction ", comparing the three kinds of analysis method, obtained by analysis" the percentage of infected cells is the most accurate matching area subtraction "among the three methods.
In this paper, the growth time, cell cycle effects on cell activity and virus infection were also studied. The growth time effect on cell activity and virus infection is acquired by lag phase, logarithmic growth phase and stationary phase cells of three different periods of cell activity by MTT method and the strength of the relationship. Infected with the virus, the virus replication ability to determine the three different periods of infected cells were the strength. The final conclusions are as follows: (1) the study period of delay cell activity by MTT method, the strength of the relationship between the three stages of the logarithmic growth phase and stationary phase, relationship of cell activity is: logarithmic growth during the lag phase stable phase. > > (2) delay, viral replication capacity of the three different periods of logarithmic growth phase and stationary phase cells were infected by the strength of the relationship is: the logarithmic growth phase, lag phase, stability stage
Effect of cell cycle on cell activity and viral infection, first of all is the synchronization of the cells with nocodazole, and then collected different initial cell cell cycle (G1 phase cells, the largest proportion of the largest proportion of the S phase cells and G_2/M cells. The largest proportion of) experiment to determine the cell activity of three periods. The strength of the relationship for the use of MTT method: G_1 > S > G_2/M period, replication capacity three the infected cells were in the strength of the relationship: G_1 > S > G_2/M. The last period of virus infection on the cell cycle have significant effect, when Sf9 cells were infected by AcMNPV virus after the cell cycle will eventually block and accumulated in the G_2/M phase.

【學(xué)位授予單位】：武漢工程大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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