本文關(guān)鍵詞： 氣腫疽 CctA基因 核酸疫苗 動物免疫　出處：《延邊大學》2017年碩士論文　論文類型：學位論文

【摘要】：氣腫疽(Clostridium chauvoei)是一種由專性厭氧菌氣腫疽梭菌引起的牛、羊等急性、敗血性傳染病。常見的病理特征分別為肌群部位有炎性癥狀、氣性腫脹,觸壓時產(chǎn)生捻發(fā)音,并且具有地區(qū)流行性、季節(jié)性等特點。此病夏季多發(fā),多見于潮濕的山谷牧場和沼澤地區(qū)。延邊朝鮮族自治州位于吉林省東側(cè),山區(qū)叢林茂密多雨,近些年牛氣腫疽的發(fā)病率相對較高,給當?shù)氐男竽翗I(yè)和養(yǎng)殖業(yè)發(fā)展影響巨大,并且對當?shù)鼐用竦恼Ｉ钜伯a(chǎn)生了極大的威脅。為了控制延邊地區(qū)氣腫疽病的發(fā)病率,制備安全且有效的氣腫疽核酸疫苗是非常必要的,本試驗的建立基礎(chǔ)是根據(jù)NCBI上氣腫疽梭菌細胞毒素A(CctA)的基因序列設(shè)計出一對特異性引物。將PCR擴增的CctA基因通過PCR擴增后克隆到pMD19-T simple載體,分析通過正確測序的CctA基因序列,再次克隆至pcDNA3.1-1真核表達載體,并且將其轉(zhuǎn)染到Vero細胞,用來確定CctA基因是否表達的方法分別為間接免疫熒光和免疫印跡兩種方法。測定表達質(zhì)粒濃度后進行小鼠免疫試驗,使用間接ELISA測定小鼠IgG1、IgG2a抗體水平,用ELISA試劑盒測定IL-4和IFN-γ細胞因子水平。研究結(jié)果顯示,CctA基因通過擴增后為519 bp,同源性比對氣腫疽梭菌ATCC 10092菌株和測序后得到的CctA基因核苷酸和氨基酸序列,存在3處點突變,與其他6株菌氨基酸序列比較存在2個位點突變;這些氨基酸序列的同源性為98.8%,核苷酸序列的同源性為99.4%。pcDNA3.1-CctA組通過轉(zhuǎn)染后的細胞存在綠色熒光的現(xiàn)象,同時pcDNA3.1-CctA重組質(zhì)粒表達的產(chǎn)物大小約是19 kD,說明Vero細胞中的pcDNA3.1-CctA質(zhì)粒成功表達了 CctA蛋白。BALB/c小鼠免疫學試驗表明,三免后,pcDNA3.1-cctA組和蛋白組的IgG1和IgG2a極顯著高于PBS組和pcDNA3.1組(P0.01),PBS組和pcDNA3.1組的IgG1和IgG2a水平無顯著性差異(P0.05);蛋白組細胞因子IFN-y和IL-4的表達水平均極顯著高于PBS組和pcDNA3.1組,顯著低于pcDNA3.1-CctA組(P0.05),在一定程度上更好的增加了小鼠的免疫水平。本試驗預測和分析了延邊株氣腫疽的信息,構(gòu)建了氣腫疽pcDNA3.1-CctA質(zhì)粒并成功表達,且能夠成功誘導小鼠產(chǎn)生體液免疫應答和細胞免疫應答。
[Abstract]:Clostridium chauvoeii is an acute condition caused by Clostridium chauvoeii, caused by Clostridium chauvoeii, which is caused by Clostridium chauvoeii, which is caused by Clostridium chauvoeii. Septic infectious disease. The common pathological characteristics are inflammatory symptoms in muscle group, swelling of breath, twisting when touching pressure, and regional epidemic, seasonal and so on. The disease is more frequent in summer. Yanbian Korean Autonomous Prefecture is located on the east side of Jilin Province, the mountain area is dense and rainy, the incidence of cattle emphysema is relatively high in recent years. In order to control the incidence of emphysema in Yanbian area, it has a great impact on the development of local animal husbandry and aquaculture industry, and also has a great threat to the normal life of local residents. It is necessary to prepare a safe and effective nucleic acid vaccine for emphysema. The basis of this experiment is based on the cytotoxin of Clostridium emphysema on NCBI. The CctA gene amplified by PCR was amplified by PCR and cloned into pMD19-T simple vector. The CctA gene was sequenced and cloned into pcDNA3.1-1 eukaryotic expression vector and transfected into Vero cells. Indirect immunofluorescence and Western blotting were used to determine the expression of CctA gene in mice. The level of IgG2a antibody in mice was determined by indirect ELISA, and the levels of IL-4 and IFN- 緯 cytokines were measured by ELISA kit. The CctA gene was 519 BP after amplification. The homology was compared with the nucleotide and amino acid sequences of CctA gene obtained from ATCC 10092 strain of Clostridium emphysema. There were 3 point mutations and 2 loci mutations compared with the other 6 strains. The homology of these amino acid sequences is 98.8 and the homology of nucleotide sequence is 99.4. PcDNA3.1-CctA group has the phenomenon of green fluorescence after transfection. At the same time, the product size of pcDNA3.1-CctA recombinant plasmid was about 19 KD. The results showed that the pcDNA3.1-CctA plasmid in Vero cells successfully expressed CctA protein. BALB / c mice immunological test showed that after three immunizations. The IgG1 and IgG2a of pcDNA3.1-cctA group and protein group were significantly higher than that of PBS group and pcDNA3.1 group (P 0.01). There was no significant difference in the levels of IgG1 and IgG2a between PBS group and pcDNA3.1 group (P 0.05). The expression levels of cytokines IFN-y and IL-4 in protein group were significantly higher than those in PBS group and pcDNA3.1 group, and significantly lower than that in pcDNA3.1-CctA group (P0.05). This experiment predicted and analyzed the information of emphysema of Yanbian strain, constructed the pcDNA3.1-CctA plasmid of emphysema and expressed it successfully. And it can induce humoral immune response and cellular immune response in mice successfully.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.23

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本文編號：1445047