表达非洲猪瘟病毒p72抗原重组新城疫病毒的拯救及其免疫效果评价

发布时间：2018-03-11 17:32

本文选题：非洲猪瘟病毒　切入点：p72　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：非洲猪瘟是由非洲猪瘟病毒引起的猪的一种出血性、高致死性疾病。自1921年在肯尼亚被发现以来,到目前为止已经传播到很多国家。近年来在高加索地区的流行尤其频繁,我国与俄罗斯接壤,边境地区的软蜱和野猪的存在使非洲猪瘟传入我国的风险加大。有关非洲猪瘟疫苗的研究很多,但至今仍没有商业化的疫苗可用。关于非洲猪瘟的免疫保护机制目前还不清楚,体液和细胞免疫在其中发挥的作用也各有说法。本研究利用反向遗传操作技术,以新城疫病毒作为载体,构建了表达非洲猪瘟病毒p72蛋白的重组病毒疫苗,并且在小鼠模型上从体液和细胞免疫反应两方面对其进行了评价。本研究首先在本实验室改造的新城疫病毒致弱株MG7的P和M基因之间引入Pme I酶切位点,将非洲猪瘟病毒的p72基因插入到MG7基因组的P和M基因之间。将全长克隆和辅助质粒通过脂质体转染法转染至重组痘病毒处理后的BHK-21细胞拯救出重组病毒,命名为r NDV/p72。从四个方面对重组病毒的生物学特性进行了鉴定:通过免疫印迹实验鉴定了非洲猪瘟病毒p72蛋白的表达;连续传代10次后经测序证实了p72基因稳定遗传;比较了该重组毒和亲本毒对鸡胚、雏鸡和小鼠的致病性以及在BHK-21细胞上的生长特性,外源基因的插入使得病毒的毒力降低,其在细胞上的生长能力也略有降低。进一步利用BALB/c小鼠模型对重组病毒的免疫效果进行了评价。将所述重组病毒r NDV/p72经肌肉注射免疫BALB/c小鼠,同时设亲本毒对照组和PBS对照组;每隔两周进行一次免疫,共免疫四次。在第三次和第四次免疫一周后,通过酶联免疫吸附试验(ELISA)检测小鼠血清中诱导产生的p72特异性Ig G抗体以及Ig G亚型Ig G1和Ig G2a抗体的含量。四免二周后,取小鼠的脾脏分离淋巴细胞通过ELISOPT实验检测细胞因子IL-4和IFN-?分泌细胞的数量,同时利用细胞增殖实验评价其细胞免疫反应的强弱。ELISA结果显示三次及四次免疫后重组病毒免疫组p72特异性的Ig G抗体都可检测到,与对照组相比差异显著(p0.05);且四免后的重组病毒免疫组Ig G抗体含量要高于第三次免疫,相比差异显著(p0.05);Ig G1抗体的含量比Ig G2a高,Th2/Th1比率为6左右。IL-4和IFN-?分泌细胞数及细胞增殖幅度检测结果均为:重组病毒r NDV/p72免疫组高于亲本毒对照组,统计学分析,存在显著性差异(p0.05)。综上,本研究获得的表达非洲猪瘟病毒p72基因的新城疫病毒活载体疫苗(r NDV/p72)不仅可以诱导小鼠的体液免疫反应,同时还可以很好地激发小鼠的细胞免疫反应,为非洲猪瘟的防治提供一种候选疫苗。
[Abstract]:African swine fever is a hemorrhagic, highly lethal disease in pigs caused by the African swine fever virus. It has spread to many countries since it was discovered in Kenya in 1921. The epidemic has been particularly frequent in the Caucasus in recent years. China borders with Russia. The presence of soft ticks and wild boars in the border areas increases the risk of African swine fever being introduced into our country. There is a lot of research on African swine fever vaccines. However, there is no commercial vaccine available. The immune protection mechanism of African swine fever is not clear, and the roles of humoral and cellular immunity are also discussed. The recombinant virus vaccine expressing p72 protein of African swine fever virus was constructed by using Newcastle disease virus as vector. In this study, humoral and cellular immunoreaction were evaluated in mouse models. In this study, Pme I restriction sites were introduced between P and M genes of Newcastle disease virus (NDV) weakened strain MG7 in our laboratory. The p72 gene of African swine fever virus was inserted into the P and M genes of MG7 genome. The full-length clone and auxiliary plasmid were transfected into BHK-21 cells treated with recombinant poxvirus by liposome transfection to save the recombinant virus. It was named r NDV / p72.The biological characteristics of the recombinant virus were identified from four aspects: the expression of p72 protein of African classical swine fever virus was identified by Western blotting, and the stable inheritance of p72 gene was confirmed by sequencing after 10 successive passages. The pathogenicity of the recombinant virus and the parental virus on chicken embryo, chicks and mice and their growth characteristics on BHK-21 cells were compared. The virulence of the virus was reduced by inserting foreign gene. The immune effect of the recombinant virus was evaluated by using BALB/c mouse model. The recombinant virus r NDV/p72 was injected intramuscularly into BALB/c mice, and the parental control group and PBS control group were set up. Once every two weeks, four times. One week after the third and 4th immunizations, Enzyme linked immunosorbent assay (Elisa) was used to detect the levels of p72 specific IgG antibody and IgG G 1 and IgG G 2a antibody induced in serum of mice. The cytokines IL-4 and IFN were detected by ELISOPT assay in spleen isolated lymphocytes of mice. The number of secreting cells, and the results of Elisa for evaluating the cellular immune response by cell proliferation test. The results showed that the specific IgG antibody against p72 could be detected in the recombinant virus immunized group after three and four times immunization. Compared with the control group, the level of IgG antibody in the recombinant virus immunized group was higher than that in the third immunization group, and the level of IgG G1 antibody was significantly higher than that in the Ig G2a group. The ratio of Th2 / Th1 was about 6. IL-4 and IFN? The results of secretory cell number and cell proliferation were as follows: r NDV/p72 immunized group was higher than parent virus control group, and there was significant difference between the two groups (p 0.05). In this study, Newcastle disease virus (NDV / p72) vaccine expressing p72 gene of African swine fever virus (ACSV) could not only induce humoral immune response in mice, but also stimulate the cellular immune response of mice. To provide a candidate vaccine for the prevention and control of African swine fever.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

【参考文献】