AcMNPV感染悬浮培养Sf9细胞动力学参数的测定及影响因素的研究

发布时间：2018-04-17 05:35

本文选题：杆状病毒 + 流式细胞仪　；参考：《武汉工程大学》2012年硕士论文

【摘要】：杆状病毒表达载体系统作为最具发展前景和应用价值的表达载体系统之一，被广泛用于重组蛋白的生产、疫苗的研制以及制备生物杀虫剂。但由于杆状病毒感染过程十分复杂，现阶段又没有一个准确的、有效的感染动力学数学模型来模拟这一过程，从而给工业化生产带来了诸多不便。建立一个准确的病毒感染动力学模型，需要精确测定这一过程中的各项参数。而细胞周期与细胞生长时间对病毒复制能力也有着非常重要的影响。建立准确的病毒感染动力学模型，必需把这两个因素加入到模型中，因此本论文也对这两个因素对病毒复制能力的影响做了较为深入的研究。本论文用SYBR Green Ⅰ荧光染料对自由病毒DNA进行染色；用抗GP64蛋白的单克隆抗体（AcV1）结合自由病毒粒子，然后用荧光二抗标记AcV1从而间接标记感染细胞。这样被标记上荧光的病毒与细胞就能用流式细胞仪精确测定病毒滴度和感染细胞百分率。最终得到如下结论：（1）通过对终点稀释法和流式检测法的比较，得到大约5个BV病毒粒子等于1个TCID_(50)感染单位。（2）通过对细胞吸附病毒过程中病毒滴度的减少量和细胞的总量之间的关系得出：一个Sf9细胞的饱和病毒吸附量为50个BV，那么当MOI值大于50BVs/cell时就为高感染。（3）通过对病毒滴度的变化关系得出：细胞吸附病毒的过程要持续6h；在感染后3h，，细胞胞吞病毒的速率开始大于细胞吸附病毒的速率。（4）通过对“阴性最高荧光强度分界法”、“阴性曲线与阳性曲线交叉法”和“匹配面积相减法”，这三种分析方法的比较，得出在这三种分析方法中用“匹配面积相减法”来分析感染细胞的百分率是最为准确的。本论文对生长时间、细胞周期对细胞活性和病毒感染能力的影响也进行了研究。生长时间对细胞活性和病毒感染能力的影响是通过采集延滞期、对数生长期和稳定期的细胞，用MTT法研究三个不同时期的细胞活性的强弱关系，并用病毒感染，来确定三个不同时期的细胞被感染后的病毒复制能力的强弱。最终得到如下结论：（1）用MTT法研究延滞期、对数生长期和稳定期这三个不同时期的细胞活性强弱关系，得到细胞活性的关系是：对数生长期＞延滞期＞稳定期。（2）延滞期、对数生长期和稳定期这三个不同时期的细胞被感染后的病毒复制能力的强弱关系为为：对数生长期＞延滞期＞稳定期细胞周期对细胞活性和病毒感染能力的影响，首先是用诺考达唑对细胞进行同步化，然后采集初始细胞周期分布不同的细胞（G1期比例最大的细胞、S期比例最大的细胞和G_2/M期比例最大的细胞）。实验用MTT法确定三个期的细胞活性的强弱关系为：G_1期＞S期＞G_2/M期，得到三个不同期的细胞被感染后的病毒复制能力的强弱关系为：G_1期＞S期＞G_2/M期。最后病毒感染对细胞周期也有着显著的影响，当Sf9细胞被AcMNPV病毒感染后，细胞周期会最终阻断并累积在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.

【学位授予单位】：武汉工程大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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