本文選題：弓形蟲 + ROP7　； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：弓形蟲病是一種呈世界范圍流行的人獸共患寄生蟲病。弓形蟲病嚴(yán)重影響神經(jīng)系統(tǒng),眼部病變,新生兒全身性疾病,尤其是免疫系統(tǒng)薄弱的人。而免疫功能正常的成年人,感染通常無明顯癥狀,一般為隱性感染。當(dāng)機(jī)體免疫力受到損傷,弓形蟲隱性感染被激活,引起廣泛的組織損傷和嚴(yán)重的病變,也是艾滋病患者死亡的主要原因。弓形蟲不但危害人類健康,也嚴(yán)重的影響著畜禽牧業(yè)的發(fā)展,造成嚴(yán)重的經(jīng)濟(jì)損失。目前關(guān)于弓形蟲病尚無良好的藥物以及可實(shí)際商業(yè)化生產(chǎn)應(yīng)用的疫苗,故尋找有效預(yù)防弓形蟲病的疫苗是當(dāng)前研究的方向。弓形蟲頂端復(fù)合體—棒狀體,在入侵宿主細(xì)胞與機(jī)體感染起著重要作用,其分泌的棒狀體蛋白ROP7與弓形蟲入侵宿主細(xì)胞有關(guān),其在納蟲空泡上的拓?fù)浣Y(jié)構(gòu)還未確定,抗原表位及對宿主是否具有免疫保護(hù)性也不清楚。(1)本研究通過生物信息學(xué)技術(shù)對弓形蟲棒狀體蛋白ROP7的理化性質(zhì)、是否含有信號肽和跨膜結(jié)構(gòu)域、可溶性、二級結(jié)構(gòu)及抗原表位等進(jìn)行了預(yù)測,結(jié)果發(fā)現(xiàn)弓形蟲RH株棒狀體蛋白ROP7基因全長為1728bp,編碼575個(gè)氨基酸,N-端有一1-33位氨基酸的信號肽序列,12-34位氨基酸的跨膜結(jié)構(gòu)域,具有多個(gè)翻譯后修飾位點(diǎn),多個(gè)抗原表位,這在理論上支持了弓形蟲棒狀體蛋白ROP7具有潛在的免疫源性,有成為候選疫苗基因的潛能。(2)根據(jù)生物信息軟件的在線預(yù)測,將弓形蟲RH株棒狀體蛋白ROP7基因信號肽部位去掉,從余下的序列中截取561bp進(jìn)行PCR擴(kuò)增,將擴(kuò)增的目的片段回收純化后,克隆到pMD18-T載體,重組質(zhì)粒pMD18-T-Tg2ROP7經(jīng)NcoⅠ和NotⅠ雙酶切后,插入到pET-28a原核表達(dá)載體中,重組質(zhì)粒經(jīng)測序鑒定正確的轉(zhuǎn)入DE3感受態(tài),經(jīng)IPTG誘導(dǎo),重組蛋白ROP7進(jìn)行SDS-PAGE與Western blot分析,重組Tg2ROP7表達(dá)大小為22kDa。NI-NTA進(jìn)行純化,透析后用PEG 20000濃縮測定濃度為0.3mg/mL。(3)應(yīng)用純化的蛋白免疫小鼠,制備Tg2ROP7鼠源性多克隆抗體,ELISA檢測其效價(jià)為1∶6400,并將其成功應(yīng)用于弓形蟲Tg2ROP7在弓形蟲速殖子偏頂端的免疫熒光定位試驗(yàn)。本研究不但在理論上證明弓形蟲棒狀體蛋白ROP7具有抗原性,在技術(shù)上證明其作為弓形蟲病核酸疫苗的可行性,同時(shí)還將生物信息學(xué)技術(shù)應(yīng)用于弓形蟲蛋白的預(yù)測,這都將為深入了解弓形蟲入侵宿主細(xì)胞機(jī)理、弓形蟲病疫苗的研發(fā)和弓形蟲診斷試劑盒的研制奠定基礎(chǔ)。
[Abstract]:Toxoplasmosis (Toxoplasma gondii) is a worldwide epidemic zoonotic parasitic disease.Toxoplasma gondii severely affects the nervous system, eye lesions, neonatal systemic diseases, especially in people with weak immune systems.In adults with normal immune function, the infection is usually asymptomatic and usually recessive.When body immunity is damaged, Toxoplasma gondii recessive infection is activated, causing extensive tissue damage and serious lesions, which is also the main cause of death in AIDS patients.Toxoplasma gondii not only harm human health, but also seriously affect the development of animal husbandry, causing serious economic losses.At present, there is no good medicine for toxoplasmosis and the vaccine can be commercialized, so it is the research direction to find a vaccine to prevent Toxoplasma gondii effectively.The rodlike body of Toxoplasma gondii apical complex plays an important role in invading host cell and body infection. The rodlike protein ROP7 secreted by Toxoplasma gondii is related to Toxoplasma gondii invading host cell, and its topological structure on the vacuole of Toxoplasma gondii has not been determined.In this study, the physicochemical properties of Toxoplasma gondii rodlike protein ROP7, whether it contains signal peptides and transmembrane domains, and the solubility of Toxoplasma gondii rodlike protein ROP7 by bioinformatics were also unclear.The secondary structure and antigen epitopes were predicted. The results showed that the ROP7 gene of RH strain of Toxoplasma gondii was 1728 BP, encoding 575 amino acids with a 1-33 amino acid signal peptide sequence and a 12-34 amino acid transmembrane domain.Having multiple post-translational modification sites, multiple antigenic epitopes, which theoretically supports the potential immunogenicity of Toxoplasma gondii coryloid protein ROP7, which has the potential to be a candidate vaccine gene, according to the online prediction of bioinformatics software.The signal peptide of ROP7 gene of RH strain of Toxoplasma gondii was removed, 561bp was intercepted from the remaining sequence for PCR amplification, the amplified target fragment was recovered and purified, then cloned into pMD18-T vector. The recombinant plasmid pMD18-T-Tg2ROP7 was digested by Nco 鈪，

本文編號：1733570