弓形虫Rhomboid基因重组卡介苗的构建及其对犬的免疫效果

发布时间：2018-05-31 11:48

本文选题：弓形虫 + Rhomboid　；参考：《吉林大学》2015年硕士论文

【摘要】：刚地弓形虫(Toxoplasma gondii)是呈世界分布的寄生性原虫。弓形虫广泛寄生于人体及其它所有温血动物体的有核细胞内，可引起严重的人兽共患寄生虫病，给人类健康和畜牧业的生产带来严重的威胁。由于在弓形虫病防治方面没有理想的药物和疫苗，所以研制安全、有效的疫苗成为预防弓形虫病的工作重点。目前弓形虫基因工程苗所候选抗原基因主要有SAG1， SAG2，P28，，P23，微线粒体抗原，侵入促进因子(PEF)，54ku膜蛋白，致密颗粒抗原等。如今Rhombiod已经成为弓形虫疫苗候选基因和化药研究靶位的研究热点。IL-2是机体免疫调节网络中发挥重要作用的细胞因子。目前，关于犬白介素2（IL-2）重组卡介苗预防弓形虫病未见报道，因此本研究通过构建犬白介素2以及弓形虫Rhomboid重组卡介苗、免疫犬，检测其临床免疫效果，为深入研究犬弓形虫疫苗奠定理论基础。犬IL-2基因的克隆及序列分析提取犬脾脏RNA，根据NCBI中Genebank报道的犬IL-2基因序列及载体中所含酶切位点设计引物。通过反转录PCR扩增出犬IL-2基因开放阅读框，并将其连接到pMD18-T载体中，测序结果经Blast比对显示PCR扩增出的465bp基因开放阅读框与原序列同源性为100%。重组质粒pMV2(3)61-IL2-Rho在卡介苗中的表达及鉴定将鉴定正确的犬IL-2基因和弓形虫Rhombiod基因一起插入到大肠杆菌-分枝杆菌穿梭表达载体和整合表达载体中，构建重组质粒pMV261-IL2-Rho和pMV361-IL2-Rho，并将其转入卡介苗中，kan+抗性筛选阳性菌落，并进行传代培养。45℃热诱导目的蛋白表达后，对表达产物进行SDS-PAGE电泳分析及免疫印迹鉴定，结果表明成功构建了含有犬IL-2和弓形虫Rhomboid基因的穿梭表达载体pMV261-IL2-Rho和整合表达载体pMV361-IL2-Rho，Rhomboid基因在结核杆菌表达系统中可以进行稳定表达，并具有良好免疫原性，为进一步研制弓形虫rBCG奠定了基础。重组卡介苗pMV2（3）61-IL2-Rho对犬弓形虫感染的免疫效果将构建成功的重组卡介苗pMV261-IL2-Rho和pMV361-IL2-Rho以及pMV261-Rho和pMV361-Rho分别经皮下免疫犬，检测体液以及细胞免疫水平。结果显示，各免疫组抗体水平随免疫次数增加而上升，重组卡介苗pMV261-IL2-Rho和pMV361-IL2-Rho免疫组与对照组差异极其显著。Th1型和Th2型细胞因子进行检测，随着免疫次数增加，各试验组血清IL-2、IFN-γ水平呈上升趋势，但PBS对照组、BCG对照组没有明显变化。检测IL-4水平发现，所有组在免疫过程中，变化不明显。免疫组TNF-α浓度变化与对照组比较差异极其显著。体温检测显示，各免疫组犬的体温较对照组变化波动小，存活时间有一定的延长，检测血液里虫体数量情况发现，重组卡介苗对弓形虫的攻击有一定的免疫效果，IL-2增强重组卡介苗的免疫效果。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a worldwide parasitic protozoa. Toxoplasma is widely parasitic in human and other nuclear cells of all other warm blooded objects. It can cause serious zoonosis and bring serious threat to human health and production of animal husbandry. There is no ideal for the prevention and treatment of toxoplasmosis. Drugs and vaccines, so a safe and effective vaccine has become a key point in the prevention of toxoplasmosis. At present, the candidate antigen genes of Toxoplasma gondii are mainly SAG1, SAG2, P28, P23, micro mitochondrial antigen, intruder promoter (PEF), 54ku membrane protein, compact particle antigen and so on. Now Rhombiod has become a vaccine candidate for Toxoplasma gondii. .IL-2 is the cytokine that plays an important role in the immune regulatory network of the organism.
At present, there is no report on the prevention of toxoplasmosis by the recombinant BCG vaccine of interleukin 2 (IL-2). Therefore, the clinical immune effect was detected by constructing the canine interleukin 2 and the recombinant BCG vaccine of Toxoplasma gondii Rhomboid, and laying a theoretical foundation for the further study of the Toxoplasma canine vaccine.
The dog's IL-2 gene was cloned and sequenced to extract the dog's spleen RNA. According to the IL-2 gene sequence of the dog in NCBI and the primers in the carrier, the open reading frame of the dog IL-2 gene was amplified by reverse transcriptional PCR and connected to the pMD18-T vector, and the 465bp gene amplified by PCR was sequenced by Blast comparison. The homology of the open reading frame and the original sequence is 100%.
The expression and identification of recombinant plasmid pMV2 (3) 61-IL2-Rho in Bacillus Calmette Guerin will identify the correct dog IL-2 gene and Toxoplasma gondii Rhombiod gene into the E. coli - Mycobacterium tumefaciens shuttle expression vector and integrated expression vector, construct the recombinant plasmid pMV261-IL2-Rho and pMV361-IL2-Rho, and transfer them into BCG, kan+ resistance screening Yang. The expression products were analyzed by SDS-PAGE electrophoresis and identification by immunoblotting. The results showed that the shuttle expression vector pMV261-IL2-Rho containing IL-2 and Toxoplasma gondii Rhomboid gene, pMV261-IL2-Rho and integrated expression vector pMV361-IL2-Rho, and Rhomboid gene in Mycobacterium tuberculosis were successfully constructed. The expression system can be stable expressed and has good immunogenicity, which laid the foundation for further development of Toxoplasma gondii rBCG.
The immune effect of recombinant Bacillus Calmette Guerin pMV2 (3) 61-IL2-Rho on Toxoplasma canine infection, the successful recombinant Bacillus Calmette Guerin pMV261-IL2-Rho and pMV361-IL2-Rho, and pMV261-Rho and pMV361-Rho were immunized by subcutaneous immunization dogs respectively, and the body fluid and cellular immunity level were detected. The results showed that the antibody level of each immune group increased with the increase of the number of immune times, and the recombinant antibody level was increased. The difference between the pMV261-IL2-Rho and pMV361-IL2-Rho immune groups of BCG and the control group was significantly different between the.Th1 and Th2 type cytokines. With the increase of the number of immunization, the level of IL-2 and IFN- gamma in the serum of each test group increased, but there was no significant change in the PBS control group and the BCG control group. The detection IL-4 levels found that all groups were changed during the immune process. The change of TNF- alpha concentration in the immune group was significantly different from that in the control group. The temperature test showed that the body temperature of the dogs in each immune group was smaller than the control group, the survival time was prolonged, the number of worms in the blood was detected, the recombinant Bacillus Calmette vaccine had some immune effect on the attack of Toxoplasma, and the IL-2 enhanced the recombinant card. The immune effect of mesoporous seedlings.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.292

【参考文献】