牛气肿疽CctA基因核酸疫苗的制备及免疫效果评价

发布时间：2018-01-19 18:34

本文关键词： 气肿疽 CctA基因核酸疫苗动物免疫　出处：《延边大学》2017年硕士论文　论文类型：学位论文

【摘要】：气肿疽(Clostridium chauvoei)是一种由专性厌氧菌气肿疽梭菌引起的牛、羊等急性、败血性传染病。常见的病理特征分别为肌群部位有炎性症状、气性肿胀,触压时产生捻发音,并且具有地区流行性、季节性等特点。此病夏季多发,多见于潮湿的山谷牧场和沼泽地区。延边朝鲜族自治州位于吉林省东侧,山区丛林茂密多雨,近些年牛气肿疽的发病率相对较高,给当地的畜牧业和养殖业发展影响巨大,并且对当地居民的正常生活也产生了极大的威胁。为了控制延边地区气肿疽病的发病率,制备安全且有效的气肿疽核酸疫苗是非常必要的,本试验的建立基础是根据NCBI上气肿疽梭菌细胞毒素A(CctA)的基因序列设计出一对特异性引物。将PCR扩增的CctA基因通过PCR扩增后克隆到pMD19-T simple载体,分析通过正确测序的CctA基因序列,再次克隆至pcDNA3.1-1真核表达载体,并且将其转染到Vero细胞,用来确定CctA基因是否表达的方法分别为间接免疫荧光和免疫印迹两种方法。测定表达质粒浓度后进行小鼠免疫试验,使用间接ELISA测定小鼠IgG1、IgG2a抗体水平,用ELISA试剂盒测定IL-4和IFN-γ细胞因子水平。研究结果显示,CctA基因通过扩增后为519 bp,同源性比对气肿疽梭菌ATCC 10092菌株和测序后得到的CctA基因核苷酸和氨基酸序列,存在3处点突变,与其他6株菌氨基酸序列比较存在2个位点突变;这些氨基酸序列的同源性为98.8%,核苷酸序列的同源性为99.4%。pcDNA3.1-CctA组通过转染后的细胞存在绿色荧光的现象,同时pcDNA3.1-CctA重组质粒表达的产物大小约是19 kD,说明Vero细胞中的pcDNA3.1-CctA质粒成功表达了 CctA蛋白。BALB/c小鼠免疫学试验表明,三免后,pcDNA3.1-cctA组和蛋白组的IgG1和IgG2a极显著高于PBS组和pcDNA3.1组(P0.01),PBS组和pcDNA3.1组的IgG1和IgG2a水平无显著性差异(P0.05);蛋白组细胞因子IFN-y和IL-4的表达水平均极显著高于PBS组和pcDNA3.1组,显著低于pcDNA3.1-CctA组(P0.05),在一定程度上更好的增加了小鼠的免疫水平。本试验预测和分析了延边株气肿疽的信息,构建了气肿疽pcDNA3.1-CctA质粒并成功表达,且能够成功诱导小鼠产生体液免疫应答和细胞免疫应答。
[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- 纬 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.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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