發(fā)布時(shí)間：2018-05-31 11:48

  本文選題：弓形蟲 + Rhomboid　； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：剛地弓形蟲(Toxoplasma gondii)是呈世界分布的寄生性原蟲。弓形蟲廣泛寄生于人體及其它所有溫血?jiǎng)游矬w的有核細(xì)胞內(nèi)，可引起嚴(yán)重的人獸共患寄生蟲病，給人類健康和畜牧業(yè)的生產(chǎn)帶來嚴(yán)重的威脅。由于在弓形蟲病防治方面沒有理想的藥物和疫苗，所以研制安全、有效的疫苗成為預(yù)防弓形蟲病的工作重點(diǎn)。目前弓形蟲基因工程苗所候選抗原基因主要有SAG1， SAG2，P28，，P23，微線粒體抗原，侵入促進(jìn)因子(PEF)，54ku膜蛋白，致密顆�？乖取Ｈ缃馬hombiod已經(jīng)成為弓形蟲疫苗候選基因和化藥研究靶位的研究熱點(diǎn)。IL-2是機(jī)體免疫調(diào)節(jié)網(wǎng)絡(luò)中發(fā)揮重要作用的細(xì)胞因子。 目前，關(guān)于犬白介素2（IL-2）重組卡介苗預(yù)防弓形蟲病未見報(bào)道，因此本研究通過構(gòu)建犬白介素2以及弓形蟲Rhomboid重組卡介苗、免疫犬，檢測其臨床免疫效果，為深入研究犬弓形蟲疫苗奠定理論基礎(chǔ)。 犬IL-2基因的克隆及序列分析提取犬脾臟RNA，根據(jù)NCBI中Genebank報(bào)道的犬IL-2基因序列及載體中所含酶切位點(diǎn)設(shè)計(jì)引物。通過反轉(zhuǎn)錄PCR擴(kuò)增出犬IL-2基因開放閱讀框，并將其連接到pMD18-T載體中，測序結(jié)果經(jīng)Blast比對顯示PCR擴(kuò)增出的465bp基因開放閱讀框與原序列同源性為100%。 重組質(zhì)粒pMV2(3)61-IL2-Rho在卡介苗中的表達(dá)及鑒定將鑒定正確的犬IL-2基因和弓形蟲Rhombiod基因一起插入到大腸桿菌-分枝桿菌穿梭表達(dá)載體和整合表達(dá)載體中，構(gòu)建重組質(zhì)粒pMV261-IL2-Rho和pMV361-IL2-Rho，并將其轉(zhuǎn)入卡介苗中，kan+抗性篩選陽性菌落，并進(jìn)行傳代培養(yǎng)。45℃熱誘導(dǎo)目的蛋白表達(dá)后，對表達(dá)產(chǎn)物進(jìn)行SDS-PAGE電泳分析及免疫印跡鑒定，結(jié)果表明成功構(gòu)建了含有犬IL-2和弓形蟲Rhomboid基因的穿梭表達(dá)載體pMV261-IL2-Rho和整合表達(dá)載體pMV361-IL2-Rho，Rhomboid基因在結(jié)核桿菌表達(dá)系統(tǒng)中可以進(jìn)行穩(wěn)定表達(dá)，并具有良好免疫原性，為進(jìn)一步研制弓形蟲rBCG奠定了基礎(chǔ)。 重組卡介苗pMV2（3）61-IL2-Rho對犬弓形蟲感染的免疫效果將構(gòu)建成功的重組卡介苗pMV261-IL2-Rho和pMV361-IL2-Rho以及pMV261-Rho和pMV361-Rho分別經(jīng)皮下免疫犬，檢測體液以及細(xì)胞免疫水平。結(jié)果顯示，各免疫組抗體水平隨免疫次數(shù)增加而上升，重組卡介苗pMV261-IL2-Rho和pMV361-IL2-Rho免疫組與對照組差異極其顯著。Th1型和Th2型細(xì)胞因子進(jìn)行檢測，隨著免疫次數(shù)增加，各試驗(yàn)組血清IL-2、IFN-γ水平呈上升趨勢，但PBS對照組、BCG對照組沒有明顯變化。檢測IL-4水平發(fā)現(xiàn)，所有組在免疫過程中，變化不明顯。免疫組TNF-α濃度變化與對照組比較差異極其顯著。體溫檢測顯示，各免疫組犬的體溫較對照組變化波動小，存活時(shí)間有一定的延長，檢測血液里蟲體數(shù)量情況發(fā)現(xiàn)，重組卡介苗對弓形蟲的攻擊有一定的免疫效果，IL-2增強(qiáng)重組卡介苗的免疫效果。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.292

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