流感病毒裂解疫苗工艺研究

发布时间：2018-07-14 12:10

【摘要】： 流感病毒具有高度的传染性,能引起急性呼吸道传染疾病,流行特点是在短时间内突然爆发,迅速蔓延,造成不同程度的流行,包括世界大流行、局部流行暴发及扩散。其发病率极高,同时伴有很高的死亡率。目前尚无特效药,接种流感疫苗是被公认的预防流感发生与传播的最佳方法,用鸡胚作为病毒生长介质也是目前流感疫苗生产最常用的培养基质。我们首先将来源于WHO流感参考和研究合作中心的H1N1、H3N2、B三种不同型别的流感病毒毒株分别接种于SPF级鸡胚,经培养后,收获其尿囊液,制备主代和工作种子批。将工作种子批接种来源于封闭式房舍内饲养的健康鸡群鸡胚进行大规模培养,收获病毒液,然后经超滤浓缩、纯化、裂解、灭活、二次纯化等步骤,获得三个型别的单价病毒液。在这过程中首先明确了适宜的毒种稀释倍数和病毒收获时间,然后摸索了蔗糖密度梯度离心纯化应该收集的病毒峰,并对工艺中裂解剂以及灭活剂的选用进行了研究,确定了生产工艺。选用两种裂解剂Triton X100和Triton N101,以不同浓度对流感病毒单价纯化液作用,通过电镜进行裂解效果观察,确定最佳裂解时间,用单向免疫扩散法检测HA含量作为评价指标。结果表明:Triton X100的裂解效果较优越,0.5%～1%,Triton X100作用90min可使流感病毒裂解。我们在本次研究中,将二乙烯亚胺(Binary Ethylene Imine,简称BEI)引入制备流感病毒灭活疫苗当中,与甲醛做对照,BEI的最佳灭活时间在24～36h之间,甲醛的为3h;BEI的灭活后的免疫效果明显优于甲醛。运用RT-PCR的方法测定出病毒8个基因灭活前后的变化,表明BEI灭活病毒的同时将8个基因序列完全破坏,而甲醛破坏不完全,从分子生物学水平证明经BEI灭活的流感灭活疫苗更加安全、可靠。通过单向免疫扩散试验对两种灭活剂的灭活效果做进一步验证,发现虽然BEI灭活病毒的时间长,但是其样品中HA含量较甲醛灭活的高,但BEI能否用于人用疫苗的制备还需进一步研究进行大量的临床实验。本研究对使用鸡胚培养流感病毒制备流感裂解疫苗生产工艺的几个重要环节进行试验摸索,为将来生产工艺的进一步改进提供了研究基础。
[Abstract]:Influenza virus is highly infectious and can cause acute respiratory tract transmission disease. The epidemic characteristic is a sudden outbreak in a short period of time, rapid spread, resulting in different levels of epidemic, including the world pandemic, local outbreak and spread. Its incidence is extremely high, accompanied by a high mortality rate. At present, there is no specific drug, inoculation of influenza vaccine is recognized as the best way to prevent the occurrence and transmission of influenza, chicken embryo as the medium of virus growth is also the most commonly used culture substrate for influenza vaccine production at present. We first inoculated three different types of influenza virus strains from WHO Influenza reference and Research collaborating Center into SPF chicken embryos. After culturing, we harvested their allantoic fluid and prepared the main generation and working seed batch. Batch inoculation of working seeds from healthy chicken embryos raised in closed houses was carried out on a large scale, and virus solution was harvested, then concentrated by ultrafiltration, purified, decomposed, inactivated, re-purified and so on. Three types of monovalent virus solution were obtained. In the process, the suitable dilution times and harvest time of virus were determined, then the virus peak should be collected by sucrose density gradient centrifugation was explored, and the selection of cracking agent and inactivating agent in the process was studied. The production process was determined. Two kinds of cracking agents Triton X100 and Triton N101 were used to treat influenza virus monovalent purified solution with different concentrations. The best lysis time was determined by electron microscope, and the HA content was detected by one-way immunodiffusion. The results showed that the cleavage effect of Triton X100 was better than that of Triton X100. Triton X100 could make influenza virus lyse with Triton X100. In this study, we introduced binary Ethylene Imine (BEI) into the preparation of Influenza virus inactivated vaccine. The best inactivation time of BEI compared with formaldehyde was between 24 and 36 hours. The immune effect of the inactivated 3 h BEI of formaldehyde was obviously better than that of formaldehyde. The changes before and after the inactivation of the 8 genes of the virus were determined by RT-PCR. The results showed that the BEI inactivated virus completely destroyed the sequence of the 8 genes while the formaldehyde was not completely destroyed. It is proved that the inactivated influenza vaccine inactivated by BEI is more safe and reliable from the level of molecular biology. The inactivation effect of two inactivated agents was further verified by one-way immunodiffusion test. It was found that although BEI inactivated the virus for a long time, the HA content in the sample was higher than that in formaldehyde inactivation. However, whether BEI can be used in the preparation of human vaccine needs further research and a large number of clinical trials. In this study, several important links in the production process of influenza lytic vaccine using chicken embryo culture influenza virus were tested and explored, which provided the basis for further improvement of the production process in the future.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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