登革4型病毒新分離株和減毒株的特性研究與登革乙腦嵌合病毒構(gòu)建初探
發(fā)布時(shí)間：2018-11-25 10:29
【摘要】：一株登革病毒是否可以作為減毒疫苗候選株，，要經(jīng)過(guò)一系列的實(shí)驗(yàn)來(lái)確定。首先明確病毒的一些體外特征如空斑的特性，溫度敏感性等，然后進(jìn)行小動(dòng)物實(shí)驗(yàn)來(lái)初步確定其毒力與免疫性，如結(jié)果良好，則可做猴體試驗(yàn)，繼續(xù)觀察其毒力和免疫性，如結(jié)果良好，則可確定為一株可用的減毒疫苗候選株。而要知道此株減毒疫苗候選株是否可以作為疫苗最終還得依靠臨床試驗(yàn)來(lái)確定。 本研究中，用從云南省西雙版納蚊體分離的Ban18原株及在原代地鼠腎細(xì)胞傳代減毒的Ban18-30株病毒進(jìn)行了生物學(xué)特性和分子特征的研究。 使用小鼠腹水制備的抗DEN-4特異性抗體與兩株病毒進(jìn)行中和實(shí)驗(yàn)，結(jié)果兩株病毒均可被其特異性中和，中和指數(shù)大于或等于1000，證明兩株病毒確為DEN-4。應(yīng)用RT-PCR方法擴(kuò)增兩株的結(jié)構(gòu)蛋白C、prM、E基因和非結(jié)構(gòu)蛋白NS1基因以及5′、3′UTR部分基因并測(cè)序，以全長(zhǎng)E基因與其它國(guó)內(nèi)外的DEN-4型毒株比較構(gòu)建的系統(tǒng)進(jìn)化樹(shù)結(jié)果也證實(shí)兩株病毒確為DEN-4。 對(duì)兩株病毒進(jìn)行空斑滴定，結(jié)果兩株病毒在Vero細(xì)胞中滴度為4.25×10~6PFU／ml，病毒產(chǎn)量高，Ban18-30株空斑較Ban18原株稍小。隨后的乳鼠毒力實(shí)驗(yàn)得知Ban18-30株較Ban18原株的logLD_(50)值至少低1.7，同時(shí)腦內(nèi)攻擊幼鼠，結(jié)果Ban18-30株對(duì)幼鼠完全不致病而原株的logLD_(50)值為4.3。初步說(shuō)明Ban18-30株是減毒的。腹腔免疫接種小鼠后用原株進(jìn)行腦內(nèi)攻擊，結(jié)果兩株的免疫原性都很好，能夠抵抗大于1000LD_(50)致死劑量的攻擊，也說(shuō)明Ban18-30株在減毒的過(guò)程中仍保留了很高的免疫原性，這對(duì)減毒活疫苗來(lái)說(shuō)也是很重要的。 對(duì)兩株部分基因序列進(jìn)行對(duì)比分析，表明E155氨基酸位點(diǎn)和3′UTR的219位核苷酸的變化很可能導(dǎo)致了Ban18-30株的毒力的減弱。
[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.
【學(xué)位授予單位】：中國(guó)藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R373
本文編號(hào)：2355773
[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.
【學(xué)位授予單位】：中國(guó)藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R373
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