肠道病毒71型的层析纯化

发布时间：2018-05-14 15:49

本文选题：肠道病毒71型 + 病毒滴度　；参考：《河南师范大学》2011年硕士论文

【摘要】：手足口病是儿童流行疾病中比较常见的疾病之一,许多种肠道病毒都可以引起手足口病,近几年来在我国以肠道病毒71型(EV71)为主要的病原体。自1997年以来,EV71病毒的流行在亚太地区呈现上升的趋势,甚至引起了严重的中枢神经系统的感染或死亡,引起了人们的密切关注。由于目前还没有针对EV71病毒的特效药和疫苗,因此,研发一种能有效控制EV71病毒流行的疫苗已经势在必行。本研究的目的是为建立纯化EV71病毒工艺,为以后制备EV71病毒的疫苗提供可靠的研究数据。本研究以EV71病毒粗提液做为研究的材料,建立了凝胶过滤层析和离子交换层析相结合的纯化工艺,为EV71病毒纯度的提高提供了最佳的层析参数。首先,对Sepharose 4 Fast Flow凝胶过滤层析参数的筛选,选用不同的洗脱流速、不同的样品上样量、不同缓冲溶液的pH值和不同盐浓度对EV71病毒粗提液进行连续洗脱。结果表明凝胶过滤层析的洗脱流速、样品上样量、缓冲溶液的pH值和盐浓度对EV71病毒均能不同程度地影响EV71病毒的分粗提液的分离效果。在实验范围内,选择洗脱流速为60cm/h、样品的上样量为5%CV、缓冲溶液的pH值为7.5和盐浓度为0.1mol/L时,对EV71病毒原液的分离效果比较理想。接着选用了Q Sepharose XL阴离子胶作为层析介质来纯化EV71病毒原液。影响离子交换层析的因素有离子强度、pH、温度和流速等,本文主要探讨了离子强度和pH对离子交换制备EV71病毒的影响。先采用线性梯度洗脱法,初步筛选制备EV71病毒平衡缓冲液含氯化钠的浓度范围和洗脱缓冲液中氯化钠的浓度;然后在同一pH条件下,改变平衡缓冲液中氯化钠的含量,计算在每种平衡缓冲液下EV71病毒的效价和滴度,选择最佳的平衡缓冲液的氯化钠的含量;再在同一离子强度条件下,改变pH值,通过计算EV71病毒的效价和滴度,选择最佳pH值。最后通过正交法去验证了不同离子强度和pH值组合,从而筛选出了最佳层析参数。本实验可以看出离子强度和pH值均能不同程度地影响EV71病毒的制备。在实验范围内,平衡缓冲液中含200mmol/L氯化钠和pH为7.5为最佳组合。最后采用先凝胶过滤层析后离子交换层析相结合的方法,运用前面筛选的最佳层析参数,进行实验,得到了制备高纯度的EV71病毒的可行性,获得了满意的结果。该工艺简单、成本低、效价和比活高。
[Abstract]:Hand, foot and mouth disease (HFMD) is one of the most common diseases in children. Many kinds of enterovirus can cause HFMD. In recent years, enterovirus 71 (EV71) is the main pathogen in China. Since 1997, the prevalence of EV71 virus has been on the rise in the Asia-Pacific region, causing serious infection or death of the central nervous system. Because there is no specific drug and vaccine against EV71 virus, it is imperative to develop a vaccine that can effectively control the epidemic of EV71 virus. The purpose of this study was to establish a purified EV71 virus technology and to provide reliable data for the preparation of EV71 virus vaccine. In this study, the crude extract of EV71 virus was used as the research material, and the purification process of gel filtration chromatography and ion exchange chromatography was established, which provided the best chromatographic parameters for improving the purity of EV71 virus. Firstly, the parameters of Sepharose 4 Fast Flow gel filtration chromatography were screened. Different elution flow rate, sample amount, pH value of different buffer solution and different salt concentration were used to elucidate the crude extract of EV71 virus. The results showed that the elution rate of gel filtration chromatography, sample volume, pH value of buffer solution and salt concentration could affect the isolation of EV71 virus in different degrees. In the experimental range, when the elution flow rate is 60 cm / h, the sample amount is 5 CVS, the pH of buffer solution is 7.5 and the salt concentration is 0.1mol/L, the isolation effect of EV71 virus original solution is better. Then the Q Sepharose XL anion gel was used as the chromatography medium to purify the EV71 virus. The factors affecting ion exchange chromatography include ionic strength pH, temperature and flow rate. The effects of ion strength and pH on the preparation of EV71 virus by ion exchange were studied in this paper. The concentration range of sodium chloride in equilibrium buffer of EV71 virus and the concentration of sodium chloride in elution buffer were preliminarily screened by linear gradient elution method, and then the content of sodium chloride in equilibrium buffer was changed at the same pH. The titer and titer of EV71 virus were calculated in each equilibrium buffer, and the optimum pH value was selected by selecting the best sodium chloride content in the equilibrium buffer solution, and then changing the pH value under the same ionic strength condition. By calculating the titer and titer of EV71 virus, the optimal pH value was selected. Finally, the orthogonal method was used to verify the combination of different ionic strength and pH value, and the optimum chromatographic parameters were screened out. It can be seen that both ionic strength and pH value can affect the preparation of EV71 virus to some extent. In the range of experiment, the best combination of 200mmol/L sodium chloride and pH 7.5 was equilibrium buffer solution. Finally, the method of combining gel filtration chromatography with ion exchange chromatography and the best chromatographic parameters were used to carry out experiments. The feasibility of preparing high purity EV71 virus was obtained, and satisfactory results were obtained. The process is simple, low cost, high titer and high specific activity.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【相似文献】