【摘要】：本研究首先對IBV S1、IBDV VP2基因進(jìn)行亞克隆,并利用一段linker將IBV S1、IBDV VP2基因連接后,利用真核表達(dá)載體pVAX-1,通過基因重組分別構(gòu)建真核重組表達(dá)質(zhì)粒pVAX-IBV S1、pVAX-IBDV VP2和pVAX-IBV S1-linker-IBDV VP2。體外瞬時(shí)轉(zhuǎn)染實(shí)驗(yàn)表明,外源基因能夠在細(xì)胞內(nèi)進(jìn)行表達(dá),并且表達(dá)后的蛋白可以被相應(yīng)的IBV S1多克隆抗體和IBDV VP2多克隆抗體識別,說明了插入的外源基因具備各自的免疫反應(yīng)性,為動(dòng)物體內(nèi)試驗(yàn)提供了基礎(chǔ)。 將試驗(yàn)雞分為8組,即S1組(免疫pVAX-IBV S1真核重組質(zhì)粒)、VP2組(免疫pVAX-IBDV VP2真核重組質(zhì)粒)、SV組(免疫pVAX-IBV S1-linker-IBDV VP2真核重組質(zhì)粒)、SV18組(免疫pVAX-IBV S1真核重組質(zhì)粒和pVAX-ChIL-18真核重組質(zhì)粒)、18組(免疫pVAX-ChIL-18真核重組質(zhì)粒)、YM組(免疫IBVH120疫苗和IBDV疫苗)、PBS組(免疫PBS)、pVAX組(免疫pVAX空載體質(zhì)粒)。免疫過程中利用淋巴細(xì)胞增殖試驗(yàn)、酶聯(lián)免疫吸附試驗(yàn)、中和抗體試驗(yàn)以及利用流式細(xì)胞術(shù)檢測CD4+、CD8+T淋巴細(xì)胞動(dòng)態(tài)變化,對不同日齡的試驗(yàn)動(dòng)物進(jìn)行檢測,最后用病毒攻擊試驗(yàn)比較免疫不同質(zhì)粒組別之間保護(hù)率。 實(shí)驗(yàn)結(jié)果表明,二聯(lián)核酸疫苗對兩種病毒的攻擊有一定的保護(hù)作用,添加了ChIL-18作為免疫佐劑增強(qiáng)了二聯(lián)核酸疫苗的免疫效果,同時(shí)增加了核酸疫苗對病毒攻擊的保護(hù)力。
[Abstract]:In this study, we first subcloned the IBV S1G IBDV VP2 gene, then ligated the IBV S1 BDV VP2 gene with a piece of linker, then constructed the eukaryotic expression plasmids pVAX-IBV S1 pVAX-IBDV VP2 and pVAX-IBV S1-linker-IBDV VP2. by gene recombination using the eukaryotic expression vector pVAX-1,. Transient transfection in vitro showed that the foreign gene could be expressed in cells, and the expressed protein could be recognized by the corresponding IBV S1 polyclonal antibody and IBDV VP2 polyclonal antibody. The results showed that the inserted foreign genes had their own immunoreactivity, which provided the basis for in vivo experiments in animals. The chickens were divided into 8 groups. That is, S1 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid) and VP2 group (immunized with pVAX-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1-linker-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid). The recombinant plasmids were divided into two groups: YM group (immunized with IBVH120 vaccine and IBDV vaccine) and PBS group (immunized with PBS) and pVAX group (immunized with pVAX unloaded particles). Lymphocyte proliferation test, enzyme-linked immunosorbent assay (Elisa), neutralizing antibody test and flow cytometry were used to detect the dynamic changes of CD8 T lymphocytes in different ages. Finally, virus attack test was used to compare the protective rate among different plasmid groups. The results showed that the binucleic acid vaccine had a certain protective effect against the two viruses, and the addition of ChIL-18 as an immune adjuvant enhanced the immune effect of the vaccine and increased the protection of the DNA vaccine against the virus attack.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 于京穹;雞傳染性支氣管炎病毒S1基因和M基因二價(jià)核酸疫苗構(gòu)建與免疫效力研究[D];東北農(nóng)業(yè)大學(xué);2012年

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本文編號：2258472