發(fā)布時(shí)間：2018-03-11 17:32

  本文選題：非洲豬瘟病毒　切入點(diǎn)：p72　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：非洲豬瘟是由非洲豬瘟病毒引起的豬的一種出血性、高致死性疾病。自1921年在肯尼亞被發(fā)現(xiàn)以來,到目前為止已經(jīng)傳播到很多國家。近年來在高加索地區(qū)的流行尤其頻繁,我國與俄羅斯接壤,邊境地區(qū)的軟蜱和野豬的存在使非洲豬瘟傳入我國的風(fēng)險(xiǎn)加大。有關(guān)非洲豬瘟疫苗的研究很多,但至今仍沒有商業(yè)化的疫苗可用。關(guān)于非洲豬瘟的免疫保護(hù)機(jī)制目前還不清楚,體液和細(xì)胞免疫在其中發(fā)揮的作用也各有說法。本研究利用反向遺傳操作技術(shù),以新城疫病毒作為載體,構(gòu)建了表達(dá)非洲豬瘟病毒p72蛋白的重組病毒疫苗,并且在小鼠模型上從體液和細(xì)胞免疫反應(yīng)兩方面對(duì)其進(jìn)行了評(píng)價(jià)。本研究首先在本實(shí)驗(yàn)室改造的新城疫病毒致弱株MG7的P和M基因之間引入Pme I酶切位點(diǎn),將非洲豬瘟病毒的p72基因插入到MG7基因組的P和M基因之間。將全長克隆和輔助質(zhì)粒通過脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染至重組痘病毒處理后的BHK-21細(xì)胞拯救出重組病毒,命名為r NDV/p72。從四個(gè)方面對(duì)重組病毒的生物學(xué)特性進(jìn)行了鑒定:通過免疫印跡實(shí)驗(yàn)鑒定了非洲豬瘟病毒p72蛋白的表達(dá);連續(xù)傳代10次后經(jīng)測序證實(shí)了p72基因穩(wěn)定遺傳;比較了該重組毒和親本毒對(duì)雞胚、雛雞和小鼠的致病性以及在BHK-21細(xì)胞上的生長特性,外源基因的插入使得病毒的毒力降低,其在細(xì)胞上的生長能力也略有降低。進(jìn)一步利用BALB/c小鼠模型對(duì)重組病毒的免疫效果進(jìn)行了評(píng)價(jià)。將所述重組病毒r NDV/p72經(jīng)肌肉注射免疫BALB/c小鼠,同時(shí)設(shè)親本毒對(duì)照組和PBS對(duì)照組;每隔兩周進(jìn)行一次免疫,共免疫四次。在第三次和第四次免疫一周后,通過酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測小鼠血清中誘導(dǎo)產(chǎn)生的p72特異性Ig G抗體以及Ig G亞型Ig G1和Ig G2a抗體的含量。四免二周后,取小鼠的脾臟分離淋巴細(xì)胞通過ELISOPT實(shí)驗(yàn)檢測細(xì)胞因子IL-4和IFN-?分泌細(xì)胞的數(shù)量,同時(shí)利用細(xì)胞增殖實(shí)驗(yàn)評(píng)價(jià)其細(xì)胞免疫反應(yīng)的強(qiáng)弱。ELISA結(jié)果顯示三次及四次免疫后重組病毒免疫組p72特異性的Ig G抗體都可檢測到,與對(duì)照組相比差異顯著(p0.05);且四免后的重組病毒免疫組Ig G抗體含量要高于第三次免疫,相比差異顯著(p0.05);Ig G1抗體的含量比Ig G2a高,Th2/Th1比率為6左右。IL-4和IFN-?分泌細(xì)胞數(shù)及細(xì)胞增殖幅度檢測結(jié)果均為:重組病毒r NDV/p72免疫組高于親本毒對(duì)照組,統(tǒng)計(jì)學(xué)分析,存在顯著性差異(p0.05)。綜上,本研究獲得的表達(dá)非洲豬瘟病毒p72基因的新城疫病毒活載體疫苗(r NDV/p72)不僅可以誘導(dǎo)小鼠的體液免疫反應(yīng),同時(shí)還可以很好地激發(fā)小鼠的細(xì)胞免疫反應(yīng),為非洲豬瘟的防治提供一種候選疫苗。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 常華;花群義;段綱;;非洲豬瘟病毒的分子生物學(xué)研究進(jìn)展[J];微生物學(xué)通報(bào);2007年03期

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本文編號(hào)：1599185