H5、H1亚型多表位重组腺病毒疫苗的制备及安全性评价研究

发布时间：2018-04-01 07:12

本文选题：H5、H1亚型多表位流感重组腺病毒　切入点：生物反应器　出处：《吉林农业大学》2017年硕士论文

【摘要】：禽流感病毒(AIV)是一种人畜共患传染病的病原体,影响着全球人类的所有经济和健康问题。大多数禽流感病毒亚型很少甚至不能引起水禽患病,但家禽中爆发禽流感与死亡率高密切相关。虽然禽流感病毒很少传播给人类,该病毒经过突变或重组后具有引发危及生命的感染能力。新的流感病毒株的不断出现,使预测其表现、传播、毒力或人与人之间的传播潜力颇具挑战性。由于很难预测哪些病毒株或什么位置突变将引起下一次流感大流行,所以很难提前做好准备。疫苗接种能够起到有效预防作用,因此研究新型疫苗尤为重要。流感重组腺病毒疫苗能够刺激机体产生较高水平的体液免疫和细胞免疫,且重组腺病毒载体具有稳定性好、安全性高和宿主范围广等优点,近年来受到人们广泛的关注,具有良好的发展前景。生物反应器微载体培养技术由于其培养表面积大、易于对细胞进行实时检测和控制,在疫苗生产领域被广泛应用。阴离子交换层析具有灵敏度高、选择性好、分离速度快等优点,分子筛层析具有操作条件温和、对于高分子物质分离效果好等优点,目前这两种层析纯化方法较为常用。本实验室构建了以季节性流感H5亚型和大流行性流感H1亚型HA(HA1)基因为主,以禽流感H1、H7和H9亚型HA抗原Th表位及M1表位为表达盒的重组腺病毒疫苗。通过7L生物反应器微载体培养系统对H5、H1亚型多表位流感重组腺病毒进行大量培养,将发酵产物处理后,采用阴离子交换层析和分子筛层析进行纯化,鉴定正确后,进行安全性评价试验。生物反应器培养HEK-293细胞,经过多次优化试验,确定培养参数为:温度:37℃、pH:6.9、搅拌速率:30r/min、溶氧:50%、CO2浓度:5%。培养24h后,85%以上细胞贴壁微载体,24-36h细胞增长率较高,48h更换培养液,84h细胞总数增长了4倍,之后细胞生长呈现下降趋势。利用生物反应器微载体培养技术大量扩增H5、H1亚型多表位流感重组腺病毒,当HEK-293细胞长满微载体表面80-90%时,以5MOI接种H5、H1亚型多表位流感重组腺病毒,48h后收取病毒液,处理后测得病毒滴度为1.0×1010TCID50/mL。利用生物反应器培养的H5、H1亚型多表位流感重组腺病毒的病毒滴度为1.0×1010TCID50/mL,经过阴离子交换层析纯化和分子筛层析纯化后,病毒滴度为1.0×1010TCID50/mL,两步纯化的回收率分别为30.8%和89.3%,总回收率为27.5%,对纯化后的样品进行PCR鉴定,结果表明基因未丢失。检测260nm和280nm处吸光值OD260/OD280=1.25,符合纯度要求。将发酵和纯化后的重组腺病毒进行小鼠免疫实验研究和安全性评价。研究结果表明,经过两步纯化后的H5、H1亚型多表位流感重组腺病毒疫苗能够刺激机小鼠产生特异性抗体及细胞免疫应答,且两步纯化疫苗组的细胞因子水平高于其他疫苗组。安全性评价试验结果表明,两步纯化的H5、H1亚型多表位流感重组腺病毒疫苗的安全性良好。总之,本实验初步证明了生物反应器微载体培养系统扩增的H5、H1亚型多表位流感重组腺病毒疫苗,经阴离子交换层析和分子筛层析纯化后,能使小鼠产生体液免疫和细胞免疫,具有一定的保护性作用。
[Abstract]:Avian influenza virus (AIV) is a zoonotic pathogen of infectious diseases that affect all economic and human health problem worldwide. Most subtypes of avian influenza virus caused little or no waterfowl in poultry disease, but the outbreak of bird flu and high mortality. Although closely related to avian influenza virus rarely spread to humans, the virus after mutation or recombination has triggered life-threatening infection ability. New influenza virus strains continue to emerge, to predict its performance, communication, communication between the potential virulence or challenging people. Because it is difficult to predict what strains or what position mutation will cause the next influenza pandemic, it is to prepare in advance. Vaccination can play an effective preventive effect, so the research of new vaccines is particularly important. Influenza recombinant adenovirus vaccine can induce the humoral immune level And cellular immunity, and the recombinant adenovirus vector has good stability, high safety and wide host range and other advantages, has attracted much attention in recent years, with good prospects for development. The microcarrier bioreactor culture because of its large surface area, easy to carry out real-time detection and control of the cell, is widely used in vaccine production. Anion exchange chromatography has advantages of high sensitivity, good selectivity, separation speed, molecular sieve chromatography has the advantages of mild operating conditions, the separation effect of polymer material and so on, the two purification methods are commonly used. The laboratory was constructed with seasonal influenza H5 and influenza pandemic H1 type HA (HA1) gene mainly to avian influenza H1, H7 and H9 subtype HA Th antigen epitope and M1 epitope recombinant adenovirus vaccine expressing box. Through the 7L bioreactor culture system The system of H5, H1 subtype influenza multi epitope recombinant adenovirus were cultured in a fermentation product processing, chromatography and molecular sieve chromatography were purified by anion exchange, after correct identification, safety evaluation test. HEK-293 cell culture bioreactor, after several optimization test, determine the training parameters: temperature C: 37, pH:6.9: 30r/min, stirring rate, dissolved oxygen concentration: 50%, CO2: 5%. after 24h, more than 85% of adherent cells in microcarrier, 24-36h cells with high growth rate, 48h medium was changed, the total number of 84h cells increased 4 times, after the cell growth decreased. By using bioreactor culture a large number of techniques of H5 amplification, H1 subtype influenza multi epitope recombinant adenovirus, when HEK-293 cells were covered with micro 80-90% carrier surface, with 5MOI H5 vaccination, H1 subtype influenza multi epitope recombinant adenovirus, 48h after receiving treatment after disease poison, measured the titer of virus was 1. 0 * 1010TCID50/mL. by bioreactor culture of H5, the virus titer of H1 subtype influenza multi epitope recombinant adenovirus is 1 * 1010TCID50/mL, after screen chromatography anion exchange chromatography and molecular purification, the virus titer was 1 * 1010TCID50/mL, two step purification and recovery rate are 30.8% and 89.3%, the total recovery rate 27.5%, PCR to identify the purified samples, results showed that the gene was not lost. The detection of 260nm and 280nm absorption value of OD260/OD280=1.25, meet the requirements. The fermentation and the purity of purified recombinant adenovirus of mouse immune experiment research and safety evaluation. The results show that, after two step purification of H5 after H1. Subtypes of influenza multi epitope recombinant adenovirus vaccine can stimulate mice to produce antibody and machine specific cellular immune response, and the two step purification vaccine group and cytokine levels higher than the other group. The vaccine safety evaluation test The results show that the two step purification of H5 subtype H1 multi table safety flu recombinant adenovirus vaccine. In conclusion, this experiment demonstrated that bioreactor culture system was H5, H1 subtype influenza multi epitope recombinant adenovirus vaccine, chromatography and molecular sieve chromatography purified by anion after the exchange, the humoral and cell immunity, has a protective effect.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.5

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