弓形虫ERK7基因功能研究与蛋白结构预测

发布时间：2018-01-15 22:19

本文关键词：弓形虫ERK7基因功能研究与蛋白结构预测　出处：《黑龙江八一农垦大学》2016年博士论文　论文类型：学位论文

【摘要】：丝裂原活化蛋白激酶(MAPK)是生物体信号转导通路的重要组分,主要参与机体细胞增殖、分化和凋亡等生命活动,与应激反应、炎症和肿瘤的发生发展密切相关。MAPK家族成员众多,其中包括细胞外信号调节激酶(ERK)亚家族。弓形虫(T.gondii)内存在两种MAPK分子即TgMAPK1和TgERK7。TgMAPK1与T.gondii速殖子和缓殖子间转换有关;TgERK7分子报道很少,仅限于TgERK7基因构成等方面。为填补TgERK7基因研究空白和探究TgERK7生物学功能,我们从以下几个方面对TgERK7进行了研究。1 pVAX/TgERK7重组质粒的免疫原性分析运用DNAStar 5.0、NCBI/BLAST和Antigen Profiler Peptide Tool等生物学软件对TgERK7氨基酸序列进行系统分析,选择并确定5条候选抗原即3-14 aa,237-249 aa,346-358aa,461-472 aa和647-658 aa。经合成、偶联KLH和多次免疫家兔,成功获得五种效价高、特异性强的多克隆抗体(αA、αB、αC、αD和αE)。通过对Triton X-100通透的T.gondii GT1速殖子荧光染色发现,αA-αE均具有良好的生物学结合能力。本研究成功构建了pVAX/TgERK7真核表达质粒并评价了其免疫原性。经免疫和分析发现,pVAX/TgERK7能刺激BALB/c鼠产生低水平的细胞免疫应答,伴有一定程度的特异性抗T.gondii抗体的产生。攻虫实验结果显示,免疫鼠存活时间分别为10.8±0.2 d(GT1感染)和19.4±1.1 d(PRU感染),与未免疫对照组(pVAX I、PBS和Control)间并无差异(P0.05)。可见,pVAX/TgERK7质粒对BALB/c鼠无保护效果,尽管可刺激机体产生一定程度的体液和细胞免疫应答。2原核表达TgERK7蛋白及其免疫保护性评价本研究成功克隆并构建了原核表达载体pET/TgERK7,经体外IPTG诱导、SDS-PAGE电泳检测及蛋白纯化与定量等过程,并对TgERK7蛋白的免疫原性进行研究。结果显示,TgERK7蛋白和弗氏佐剂均能刺激BALB/c鼠产生抗体,伴有CD3e+CD4+T淋巴细胞和IFN-γ、TNF-α、IL2等多种因子升高。结果表明,TgERK7蛋白联合弗氏佐剂能够刺激BALB/c鼠产生较强的体液和细胞免疫应答;除抗A肽抗体外,其余抗体均无抗T.gondii特异性,推测可能与弗氏佐剂的使用有关。攻虫感染实验结果显示,TgERK7蛋白和弗氏佐剂联合免疫可显著延长T.gondii GT1速殖子感染BALB/c鼠的急性致死时间(13.8±3.4 d)和降低PRU脑包囊数,但对抵抗T.gondiipru株急性感染作用不明显。结果表明,tgerk7蛋白对balb/c鼠免疫保护性较好。经对比分析tgerk7蛋白和pvax/tgerk7重组质粒所引起balb/c鼠体液和细胞免疫各项指标的变化情况,发现特异性抗体αa在抵抗t.gondiigt1速殖子感染过程中起到不可忽视的重要作用。为验证上述观点,我们使用高浓度的特异性抗t.gondii抗体(αa、αb、αc、αd或αe)胞外处理gt1速殖子,并于不同时间点对其胞内增殖能力进行检测和分析;同设空白对照即未处理组。结果显示,与空白对照相比,经抗体αa处理的速殖子胞内增殖能力显著减弱,且差异程度随检测时间的延长而增大,其余各组间差异均不显著(p0.05)。结果表明,特异性抗体αa可阻碍t.gondiigt1速殖子侵染宿主细胞。3弓形虫Δtgerk7缺失株的构建与毒力分析为深入探究tgerk7基因的生物学功能,我们运用同源重组原理成功构建了重组体Δerk7-pbluescriptiisk(+),经电转t.gondiigt1速殖子和药物筛选,实现了对tgerk7转座子部分序列的成功替换。经pcr扩增、southernblotting和westernblotting鉴定发现,tgerk7基因部分缺失可导致该基因的完全沉默即最终获得了基因缺失虫株Δtgerk7。毒力实验结果显示,与gt1野生株(10.6±0.3d)相比,Δtgerk7速殖子并不能引起balb/c鼠发生死亡;有趣的是,首次感染35d后再次感染相同剂量的野生株速殖子也不能造成Δtgerk7感染鼠死亡,提示tgerk7蛋白与t.gondiigt1野生株毒力密切相关,可能参与了t.gondii速殖子致病甚至急性致死宿主的病理过程。血清抗体以及腹腔液和血清内细胞因子、no等免疫指标检测结果表明,炎性因子如ifn-γ、mcp-1和il6等的过表达是导致t.gondiigt1速殖子感染鼠急性死亡的主要原因。通过对被感染鼠腹腔中和脾组织内虫子数定量分析发现,感染7d时腹腔内Δtgerk7虫子数极显著降低,而其他各时间点和脾内检虫数均无差异(p0.05),与细胞因子检测结果基本相符。据此可认为,tgerk7基因是t.gondiigt1速殖子胞内增殖的重要调节因子,与gt1野生株毒力关系密切。4弓形虫tgerk7基因的生物学功能研究为增强实验说服力和提供重要实验参照,以puc19载体为骨架,运用特异性pcr扩增和限制酶多重酶切等分子生物学基本操作方法,成功克隆并构建了补充载体puc/sag1/cat/gra1/sag1/tgerk7/gra1。经多次电转缺失株Δtgerk7虫体和药筛,特异性pcr扩增和westernblotting鉴定以及胞内增殖能力分析,最终获得puc/tgerk7补充株,实现了TgERK7基因在ΔTgERK7缺失虫体内的再表达。T.gondii GT1野生株、ΔTgERK7缺失株和pUC/TgERK7补充株的虫斑大小以及逸出、胞内增殖、入侵、吸附和运动能力等检测和分析结果,结合TgERK7蛋白亚细胞定位结果即位于GT1速殖子顶端,发现TgERK7蛋白是T.gondii GT1速殖子入侵宿主细胞的重要作用因子,参与T.gondii速殖子的入侵过程。5弓形虫TgERK7蛋白结构预测运用PSI-PRED、DISOPRED、MEMSAT-SVM和SWISS-MODEL等蛋白结构预测软件,成功对T.gondii GT1速殖子TgERK7蛋白进行了二级和三级结构预测,并结合TgERK7基因生物学功能,推测了TgERK7蛋白在T.gondii速殖子入侵宿主细胞过程中可能存在的作用方式,即T.gondii速殖子经滑行运动靠近宿主细胞时,TgERK7蛋白因与宿主细胞表面受体结合而被激活,进而引起TgERK7蛋白分子胞内区域发生变构等反应,将接触信息跨膜传进速殖子内,引起速殖子发生一系列变化如蛋白表达、分泌或虫体变型等,为T.gondii侵染宿主细胞提供有利条件。通过评价pVAX/TgERK7重组质粒和TgERK7蛋白免疫原性,同源替换TgERK7转座子部分序列和毒力检测,构建补充株pUC/TgERK7和预测TgERK7蛋白结构等研究,发现TgERK7分子为T.gondii GT1速殖子入侵细胞的重要因子,介导T.gondii速殖子入侵宿主细胞,并提出侵染时可能存在的作用方式,填补了T.gondii ERK7基因生物学功能的研究空白,为日后深入研究TgERK7蛋白作用机制和T.gondii致病机理奠定了基础。后续研究应集中在搜寻TgERK7蛋白作用底物与解析TgERK7蛋白天然构象等方面。
[Abstract]:Mitogen activated protein kinase (MAPK) is an important biological signal transduction pathway, mainly involved in cell proliferation, differentiation and apoptosis of life activities, and stress response, inflammation and tumor occurrence and development of.MAPK is closely related to the many family members, including extracellular signal regulated kinase (ERK) subfamily of Toxoplasma gondii (T.gondii.) memory in two MAPK TgMAPK1 and TgERK7.TgMAPK1 T.gondii with molecular tachyzoite and bradyzoite conversion; TgERK7 molecule is rarely reported, only TgERK7 gene and so on. In order to fill the research blank of TgERK7 gene and explore the biological functions of TgERK7, we from the following aspects of the TgERK7 the immunogenicity of.1 pVAX/TgERK7 the recombinant plasmid was analyzed by DNAStar 5, NCBI/BLAST and Antigen Profiler Peptide Tool biology software system analysis of TgERK7 amino acid sequence, selection And to determine the 5 candidate antigen 3-14 AA, 237-249 AA, 346-358aa, 461-472 AA and 647-658 aa. by KLH synthesis, coupling and multiple immune rabbits successfully obtained five kinds of high titer, polyclonal antibody specificity (alpha A, alpha B, alpha C, alpha D alpha and E) found by T.gondii. GT1 Triton X-100 tachyzoites to transparent fluorescent staining, alpha A- alpha E has good biological binding ability. This study successfully constructed pVAX/TgERK7 eukaryotic expression plasmid and its immunogenicity was evaluated. The immune and cellular immune response analysis showed that pVAX/TgERK7 can stimulate BALB/c production in low level, with specific a certain degree of anti T.gondii antibodies. The experimental results show that the immune attack insects, the survival time of rats were 10.8 + 0.2 D (GT1 infection) and 19.4 + 1.1 D (PRU infection), and immunized control group (pVAX I, PBS and Control) had no difference (P0.05). Therefore, pVAX/TgERK7 plasmid on BAL B/c mice have no protective effect, although can stimulate the body to produce a certain degree of protein TgERK7 and its protective immunity evaluation this study successfully cloned and constructed the prokaryotic expression vector pET/TgERK7 and prokaryotic expression of humoral and cellular immune responses induced by.2 and IPTG in vitro, SDS-PAGE electrophoresis and protein purification and quantitative process, and study the immunogenicity the TgERK7 protein. The results showed that TgERK7 protein and Freund's adjuvant can stimulate BALB/c rats to produce antibodies, with CD3e+CD4+T lymphocytes and IFN- gamma, alpha TNF-, elevated IL2 and other factors. The results showed that TgERK7 protein combined with Freund's adjuvant can stimulate BALB/c induced strong humoral and cellular immune responses in resistance; A peptide antibody, no specific anti T.gondii antibody may rest, use with Freund's adjuvant. The infection of the experimental results show that TgERK7 protein and Freund's adjuvant combined immunization Could prolong the acute lethal time of BALB/c mice infected with T.gondii GT1 tachyzoites (13.8 + 3.4 d) and reduce the number of cysts of PRU brain, but the resistance of T.gondiipru strain of acute infection is not obvious. The results showed that tgerk7 protein on balb/c rat immune protection is good. Through the contrast analysis of tgerk7 protein and recombinant plasmid pvax/tgerk7 by the changes of balb/c induced humoral and cellular immune indexes, found the specific antibody against t.gondiigt1 alpha a tachyzoite infection plays an important role can not be ignored. In order to verify the above view, we use a specific anti T.gondii antibody with high concentration (alpha A, alpha B, alpha C, alpha D or alpha E) extracellular GT1 tachyzoites, and at different time points on the intracellular proliferation ability were detected and analyzed; the same blank that of untreated control. The results showed that compared with the control, the tachyzoites antibody alpha a treatment of intracellular proliferation Decreased and prolonged and the differences increase with the detection time increases, the differences between other groups were not significant (P0.05). The results showed that the specific antibody a can block the construction of alpha t.gondiigt1 tachyzoites of Toxoplasma gondii infected host cell.3 Delta tgerk7 mutant and Virulence Analysis of biological function of tgerk7 gene for further research. We use the principle of homologous recombination was successfully constructed recombinant erk7-pbluescriptiisk (+), the electric t.gondiigt1 tachyzoites and drug screening, the tgerk7 transposon sequences are successfully replaced. After PCR amplification, southernblotting and westernblotting identification, partial deletion of tgerk7 gene can lead to complete silencing of this gene is obtained gene deletion strains tgerk7. and GT1 virulence test results showed that the wild strain (10.6 + 0.3d), Delta tgerk7 tachyzoites did not cause death in balb/c mice is interesting, The same dose of wild strain tachyzoites infected again for the first time after 35d infection can cause tgerk7 infection in death, suggesting that tgerk7 protein and t.gondiigt1 wild-type virulence are closely related, may be involved in the pathogenesis of T.gondii disease or acute lethal tachyzoite host. Serum antibody and cytokines in the peritoneal fluid and serum, detection results no and immune parameters showed that inflammatory factors such as ifn- MCP-1 and IL6 gamma, the expression is the main cause of t.gondiigt1 tachyzoites infection in rats with acute death. The infection of peritoneal and splenic tissue in insect number quantitative analysis revealed that 7d tgerk7 infection significantly decreased the number of bugs in the abdominal cavity, and there were no differences in other time point and spleen (P0.05), check the number of insect are basically consistent with the cytokine test results. The tgerk7 gene is t.gondiigt1 tachyzoites as an important regulator of intracellular proliferation Study on biological functions of GT1, and wild strains closely related.4 tgerk7 gene of Toxoplasma gondii in order to enhance the persuasiveness and experiment provide important experimental reference to the pUC19 vector as the backbone, using specific PCR amplification and restriction enzyme digestion of the basic operation of multiple molecular biology methods, was successfully cloned and constructed of vector puc/sag1/cat/gra1/sag1/tgerk7/gra1. after repeated electrical turn the mutant Delta tgerk7 worms and drug screening, analysis of specific PCR amplification and identification of westernblotting and intracellular proliferation ability, puc/tgerk7 finally added strain, realized the TgERK7 gene in the expression of delta TgERK7 deletion parasite.T.gondii GT1 wild strain, TgERK7 strain and pUC/TgERK7 strain of missing worm spot size and escape cellular proliferation, invasion, adsorption and movement ability of detection and analysis results, combined with the subcellular localization of TgERK7 protein results in GT1 tachyzoites top The end, found that TgERK7 protein is an important factor of T.gondii GT1 tachyzoites invasion of host cells, participate in T.gondii tachyzoites of Toxoplasma gondii invasion process.5 TgERK7 protein structure prediction using PSI-PRED, DISOPRED, MEMSAT-SVM and SWISS-MODEL protein structure prediction software, the success of T.gondii GT1 tachyzoites of TgERK7 protein were predicted in two and three class structure, combined with the biological functions of TgERK7 gene, that the mode of action of TgERK7 protein may exist in T.gondii tachyzoites invading host cells in the process of T.gondii tachyzoites by gliding movement near the host cells, TgERK7 protein is activated by binding with the host cell surface receptors, thereby causing allosteric reaction of TgERK7 protein the cytosolic region, will contact information across the membrane into tachyzoites, caused by tachyzoites a series of changes such as protein expression, secretion or worm variant, as T.gondii provides favorable conditions for infecting host cells. Through the evaluation of the recombinant plasmid pVAX/TgERK7 and TgERK7 protein immunogenicity, homologous substitution of TgERK7 transposon sequences and partial virulence detection, construction of strains pUC/TgERK7 and TgERK7 protein structure prediction, molecular TgERK7 was found as an important factor in T.gondii GT1 tachyzoites invading cells, mediated by T.gondii tachyzoites the invasion of the host cell, and puts forward the mode of action of possible infection, fills the blank of T.gondii ERK7 gene, which lay a foundation for further study on the mechanism of TgERK7 protein and the pathogenic mechanism of T.gondii. Further research should focus on the role of protein substrates and analytical search TgERK7 TgERK7 protein natural conformation and so on.

【学位授予单位】：黑龙江八一农垦大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.7

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