登革4型病毒新分离株和减毒株的特性研究与登革乙脑嵌合病毒构建初探

发布时间：2018-11-25 10:29

【摘要】：一株登革病毒是否可以作为减毒疫苗候选株，，要经过一系列的实验来确定。首先明确病毒的一些体外特征如空斑的特性，温度敏感性等，然后进行小动物实验来初步确定其毒力与免疫性，如结果良好，则可做猴体试验，继续观察其毒力和免疫性，如结果良好，则可确定为一株可用的减毒疫苗候选株。而要知道此株减毒疫苗候选株是否可以作为疫苗最终还得依靠临床试验来确定。本研究中，用从云南省西双版纳蚊体分离的Ban18原株及在原代地鼠肾细胞传代减毒的Ban18-30株病毒进行了生物学特性和分子特征的研究。使用小鼠腹水制备的抗DEN-4特异性抗体与两株病毒进行中和实验，结果两株病毒均可被其特异性中和，中和指数大于或等于1000，证明两株病毒确为DEN-4。应用RT-PCR方法扩增两株的结构蛋白C、prM、E基因和非结构蛋白NS1基因以及5′、3′UTR部分基因并测序，以全长E基因与其它国内外的DEN-4型毒株比较构建的系统进化树结果也证实两株病毒确为DEN-4。对两株病毒进行空斑滴定，结果两株病毒在Vero细胞中滴度为4.25×10~6PFU／ml，病毒产量高，Ban18-30株空斑较Ban18原株稍小。随后的乳鼠毒力实验得知Ban18-30株较Ban18原株的logLD_(50)值至少低1.7，同时脑内攻击幼鼠，结果Ban18-30株对幼鼠完全不致病而原株的logLD_(50)值为4.3。初步说明Ban18-30株是减毒的。腹腔免疫接种小鼠后用原株进行脑内攻击，结果两株的免疫原性都很好，能够抵抗大于1000LD_(50)致死剂量的攻击，也说明Ban18-30株在减毒的过程中仍保留了很高的免疫原性，这对减毒活疫苗来说也是很重要的。对两株部分基因序列进行对比分析，表明E155氨基酸位点和3′UTR的219位核苷酸的变化很可能导致了Ban18-30株的毒力的减弱。
[Abstract]:Whether a dengue virus can be used as a candidate for attenuated vaccine is determined by a series of experiments. First, some of the virus characteristics in vitro, such as plaque characteristics, temperature sensitivity, etc., then small animal experiments were carried out to determine its virulence and immunogenicity. If the results were good, the virus could be tested in monkeys and continue to observe its virulence and immunity. If the results are good, it can be identified as a candidate for attenuated vaccine. It is up to clinical trials to determine whether the attenuated vaccine candidate can be used as a vaccine. In this study, the biological and molecular characteristics of Ban18 virus isolated from Xishuangbanna mosquito body in Yunnan Province and the attenuated Ban18-30 strain in primary hamster kidney cells were studied. The neutralization experiments were carried out with two strains of virus using the specific antibody against DEN-4 prepared in mouse ascites. The results showed that the two viruses could be neutralized by the specific neutralization index, and the neutralization index was greater than or equal to 1000. It was proved that the two strains were DEN-4.. RT-PCR method was used to amplify and sequence the two structural protein CnprMN E gene and the non structural protein NS1 gene and the partial gene of 5GN 3 UTR. The phylogenetic tree constructed by comparing the full-length E gene with other DEN-4 strains at home and abroad also confirmed that the two viruses were indeed DEN-4.. The results showed that the titer of the two viruses in Vero cells was 4.25 脳 10 ~ (6) PFU / ml, the virus yield was high, and the plaque of Ban18-30 strain was slightly smaller than that of Ban18 strain. The later virulence test showed that the logLD_ (50) value of Ban18-30 strain was at least 1.7 lower than that of Ban18 strain, and the logLD_ (50) value of the original strain was 4.3, while the logLD_ (50) value of the original strain was 4.3. The preliminary results showed that the Ban18-30 strain was attenuated. After the mice were inoculated intraperitoneally with the original strain, the immunogenicity of the two strains was very good, and the two strains could resist the attack with a lethal dose greater than 1000LD50, which also indicated that the Ban18-30 strain still retained high immunogenicity in the course of attenuating the virus. This is also important for live attenuated vaccines. The comparative analysis of the partial gene sequences of the two strains indicated that the changes of the E155 amino acid site and the 219-nucleotide of 3'UTR may lead to the weakening of the virulence of the Ban18-30 strain.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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