本文關(guān)鍵詞：弓形蟲ERK7基因功能研究與蛋白結(jié)構(gòu)預(yù)測　出處：《黑龍江八一農(nóng)墾大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 弓形蟲 TgERK7 免疫原性 亞細胞定位 基因功能 結(jié)構(gòu)預(yù)測

【摘要】：絲裂原活化蛋白激酶(MAPK)是生物體信號轉(zhuǎn)導(dǎo)通路的重要組分,主要參與機體細胞增殖、分化和凋亡等生命活動,與應(yīng)激反應(yīng)、炎癥和腫瘤的發(fā)生發(fā)展密切相關(guān)。MAPK家族成員眾多,其中包括細胞外信號調(diào)節(jié)激酶(ERK)亞家族。弓形蟲(T.gondii)內(nèi)存在兩種MAPK分子即TgMAPK1和TgERK7。TgMAPK1與T.gondii速殖子和緩殖子間轉(zhuǎn)換有關(guān);TgERK7分子報道很少,僅限于TgERK7基因構(gòu)成等方面。為填補TgERK7基因研究空白和探究TgERK7生物學(xué)功能,我們從以下幾個方面對TgERK7進行了研究。1 pVAX/TgERK7重組質(zhì)粒的免疫原性分析運用DNAStar 5.0、NCBI/BLAST和Antigen Profiler Peptide Tool等生物學(xué)軟件對TgERK7氨基酸序列進行系統(tǒng)分析,選擇并確定5條候選抗原即3-14 aa,237-249 aa,346-358aa,461-472 aa和647-658 aa。經(jīng)合成、偶聯(lián)KLH和多次免疫家兔,成功獲得五種效價高、特異性強的多克隆抗體(αA、αB、αC、αD和αE)。通過對Triton X-100通透的T.gondii GT1速殖子熒光染色發(fā)現(xiàn),αA-αE均具有良好的生物學(xué)結(jié)合能力。本研究成功構(gòu)建了pVAX/TgERK7真核表達質(zhì)粒并評價了其免疫原性。經(jīng)免疫和分析發(fā)現(xiàn),pVAX/TgERK7能刺激BALB/c鼠產(chǎn)生低水平的細胞免疫應(yīng)答,伴有一定程度的特異性抗T.gondii抗體的產(chǎn)生。攻蟲實驗結(jié)果顯示,免疫鼠存活時間分別為10.8±0.2 d(GT1感染)和19.4±1.1 d(PRU感染),與未免疫對照組(pVAX I、PBS和Control)間并無差異(P0.05)�？梢�,pVAX/TgERK7質(zhì)粒對BALB/c鼠無保護效果,盡管可刺激機體產(chǎn)生一定程度的體液和細胞免疫應(yīng)答。2原核表達TgERK7蛋白及其免疫保護性評價本研究成功克隆并構(gòu)建了原核表達載體pET/TgERK7,經(jīng)體外IPTG誘導(dǎo)、SDS-PAGE電泳檢測及蛋白純化與定量等過程,并對TgERK7蛋白的免疫原性進行研究。結(jié)果顯示,TgERK7蛋白和弗氏佐劑均能刺激BALB/c鼠產(chǎn)生抗體,伴有CD3e+CD4+T淋巴細胞和IFN-γ、TNF-α、IL2等多種因子升高。結(jié)果表明,TgERK7蛋白聯(lián)合弗氏佐劑能夠刺激BALB/c鼠產(chǎn)生較強的體液和細胞免疫應(yīng)答;除抗A肽抗體外,其余抗體均無抗T.gondii特異性,推測可能與弗氏佐劑的使用有關(guān)。攻蟲感染實驗結(jié)果顯示,TgERK7蛋白和弗氏佐劑聯(lián)合免疫可顯著延長T.gondii GT1速殖子感染BALB/c鼠的急性致死時間(13.8±3.4 d)和降低PRU腦包囊數(shù),但對抵抗T.gondiipru株急性感染作用不明顯。結(jié)果表明,tgerk7蛋白對balb/c鼠免疫保護性較好。經(jīng)對比分析tgerk7蛋白和pvax/tgerk7重組質(zhì)粒所引起balb/c鼠體液和細胞免疫各項指標的變化情況,發(fā)現(xiàn)特異性抗體αa在抵抗t.gondiigt1速殖子感染過程中起到不可忽視的重要作用。為驗證上述觀點,我們使用高濃度的特異性抗t.gondii抗體(αa、αb、αc、αd或αe)胞外處理gt1速殖子,并于不同時間點對其胞內(nèi)增殖能力進行檢測和分析;同設(shè)空白對照即未處理組。結(jié)果顯示,與空白對照相比,經(jīng)抗體αa處理的速殖子胞內(nèi)增殖能力顯著減弱,且差異程度隨檢測時間的延長而增大,其余各組間差異均不顯著(p0.05)。結(jié)果表明,特異性抗體αa可阻礙t.gondiigt1速殖子侵染宿主細胞。3弓形蟲Δtgerk7缺失株的構(gòu)建與毒力分析為深入探究tgerk7基因的生物學(xué)功能,我們運用同源重組原理成功構(gòu)建了重組體Δerk7-pbluescriptiisk(+),經(jīng)電轉(zhuǎn)t.gondiigt1速殖子和藥物篩選,實現(xiàn)了對tgerk7轉(zhuǎn)座子部分序列的成功替換。經(jīng)pcr擴增、southernblotting和westernblotting鑒定發(fā)現(xiàn),tgerk7基因部分缺失可導(dǎo)致該基因的完全沉默即最終獲得了基因缺失蟲株Δtgerk7。毒力實驗結(jié)果顯示,與gt1野生株(10.6±0.3d)相比,Δtgerk7速殖子并不能引起balb/c鼠發(fā)生死亡;有趣的是,首次感染35d后再次感染相同劑量的野生株速殖子也不能造成Δtgerk7感染鼠死亡,提示tgerk7蛋白與t.gondiigt1野生株毒力密切相關(guān),可能參與了t.gondii速殖子致病甚至急性致死宿主的病理過程。血清抗體以及腹腔液和血清內(nèi)細胞因子、no等免疫指標檢測結(jié)果表明,炎性因子如ifn-γ、mcp-1和il6等的過表達是導(dǎo)致t.gondiigt1速殖子感染鼠急性死亡的主要原因。通過對被感染鼠腹腔中和脾組織內(nèi)蟲子數(shù)定量分析發(fā)現(xiàn),感染7d時腹腔內(nèi)Δtgerk7蟲子數(shù)極顯著降低,而其他各時間點和脾內(nèi)檢蟲數(shù)均無差異(p0.05),與細胞因子檢測結(jié)果基本相符。據(jù)此可認為,tgerk7基因是t.gondiigt1速殖子胞內(nèi)增殖的重要調(diào)節(jié)因子,與gt1野生株毒力關(guān)系密切。4弓形蟲tgerk7基因的生物學(xué)功能研究為增強實驗說服力和提供重要實驗參照,以puc19載體為骨架,運用特異性pcr擴增和限制酶多重酶切等分子生物學(xué)基本操作方法,成功克隆并構(gòu)建了補充載體puc/sag1/cat/gra1/sag1/tgerk7/gra1。經(jīng)多次電轉(zhuǎn)缺失株Δtgerk7蟲體和藥篩,特異性pcr擴增和westernblotting鑒定以及胞內(nèi)增殖能力分析,最終獲得puc/tgerk7補充株,實現(xiàn)了TgERK7基因在ΔTgERK7缺失蟲體內(nèi)的再表達。T.gondii GT1野生株、ΔTgERK7缺失株和pUC/TgERK7補充株的蟲斑大小以及逸出、胞內(nèi)增殖、入侵、吸附和運動能力等檢測和分析結(jié)果,結(jié)合TgERK7蛋白亞細胞定位結(jié)果即位于GT1速殖子頂端,發(fā)現(xiàn)TgERK7蛋白是T.gondii GT1速殖子入侵宿主細胞的重要作用因子,參與T.gondii速殖子的入侵過程。5弓形蟲TgERK7蛋白結(jié)構(gòu)預(yù)測運用PSI-PRED、DISOPRED、MEMSAT-SVM和SWISS-MODEL等蛋白結(jié)構(gòu)預(yù)測軟件,成功對T.gondii GT1速殖子TgERK7蛋白進行了二級和三級結(jié)構(gòu)預(yù)測,并結(jié)合TgERK7基因生物學(xué)功能,推測了TgERK7蛋白在T.gondii速殖子入侵宿主細胞過程中可能存在的作用方式,即T.gondii速殖子經(jīng)滑行運動靠近宿主細胞時,TgERK7蛋白因與宿主細胞表面受體結(jié)合而被激活,進而引起TgERK7蛋白分子胞內(nèi)區(qū)域發(fā)生變構(gòu)等反應(yīng),將接觸信息跨膜傳進速殖子內(nèi),引起速殖子發(fā)生一系列變化如蛋白表達、分泌或蟲體變型等,為T.gondii侵染宿主細胞提供有利條件。通過評價pVAX/TgERK7重組質(zhì)粒和TgERK7蛋白免疫原性,同源替換TgERK7轉(zhuǎn)座子部分序列和毒力檢測,構(gòu)建補充株pUC/TgERK7和預(yù)測TgERK7蛋白結(jié)構(gòu)等研究,發(fā)現(xiàn)TgERK7分子為T.gondii GT1速殖子入侵細胞的重要因子,介導(dǎo)T.gondii速殖子入侵宿主細胞,并提出侵染時可能存在的作用方式,填補了T.gondii ERK7基因生物學(xué)功能的研究空白,為日后深入研究TgERK7蛋白作用機制和T.gondii致病機理奠定了基礎(chǔ)。后續(xù)研究應(yīng)集中在搜尋TgERK7蛋白作用底物與解析TgERK7蛋白天然構(gòu)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.

【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.7

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