弓形虫硬脂酰辅酶A去饱和酶的鉴定及功能研究

发布时间：2018-01-19 01:36

本文关键词： 弓形虫硬脂酰辅酶A去饱和酶条件性敲除过表达自噬　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：龚地弓形虫（Toxoplasma gondii)是专性细胞内寄生原虫,可以寄生十几乎所有温血动物和人的有核细胞。弓形虫在其寄生生活中,烬管可以从宿主摄取多种营养物质,但虫体仍具有相对完善的代谢系统,可以进行必要的分解及合成反应。弓形虫其有从头合成饱和脂肪酸的能力,但目前尚不清楚弓形虫中是否能合成不饱和脂肪酸。因此,我们进行了如下研究。已知硬脂酰辅酶A去饱和酶(stearoyl-coenzyme A desaturase, SCD)是不饱和脂肪酸合成中的关键酶。通过蛋白相似性比对发现,弓形虫中存在与疟原虫SCD相似性达54%的推定脂酰辅酶A去饱和酶基因(ToxoDB编号TGGT1_238950)。经生物信息学分析,该基因编码的蛋白质具有典型的去饱和酶结构域。通过反转录PCR从弓形虫RH株获得该基因的3129 bp全长编码序列,与TGGT1_238950勺序列完全一致。随后,对该蛋白进行了截短原核表达并制备鼠源抗体,蛋白印迹结果表明弓形虫速殖子中存在该蛋白的表达,蛋白分子量约为120 kDa,与预期相符。同时构建该基因的HA标签内源性标记虫株,通过间接免疫荧光实验,发现虫体内源性蛋白定位于内质网,与其他去饱和酶定位一致。因此,推定其为弓形虫的硬脂酰辅酶A去饱和酶,即为弓形虫SCD (TgSCD)。环丙烯脂肪酸(如苹婆酸)是SCD的抑制剂。我们选择苹婆酸以及几种与其结构相似的甲酯化物进行抗弓形虫的体外药效实验,发现它们都能不同程度抑制胞内弓形虫的增殖与释放。其中,苹婆酸抑制弓形虫的半数有效浓度最低,但对宿主细胞具有较明显的毒性作用；另一种含有甲氧基的药物(编号INT21)对弓形虫有明显抑制作用,且其在高浓度下对宿主细胞无明显毒性。其后,将上述药物进行小鼠体内药效实验,腹腔注射INT21可以使Pru株感染小鼠的脑荷虫量显著减少,但对RH株感染小鼠无明显保护作用。为了进一步研究SCD在弓形虫发育和增殖中的作刖,在弓形虫’TATi虫株中运用CRISPR/Cas9技术获得SCD的条件性敲除虫株。经噬斑实验和增殖实验发现,敲除虫体的入侵、发育和繁殖等表型无明显变化；给小鼠接种1000个速殖子,缺失SCD的虫株与TATi虫株对小鼠的毒力差异不显著,提示弓形虫速殖子阶段生长发育对SCD的依赖程度不高,可能是在SCD缺失时有其它因子替代或补偿其作用。为进一步观察SCD在虫体中功能,构建了弓形虫SCD过表达虫株。对过表达虫株的脂肪酸检测发现,虫体油酸的比例增加,证明弓形虫SCD在虫体内发挥了去饱和酶的功能。过表达虫株的体外表型观察发现,SCD过表达虫株增殖速度减慢,入侵能力降低；小鼠接种实验中,过表达虫株对小鼠的毒力低于野毒株。通过TgIF2α磷酸化和相关基因表达水平检测发现弓形虫SCD过表达虫株发生内质例应激,并且史易发生自噬和捌广。综上所述,本研究明确了弓形虫中存在SCD,且能够发挥去饱和酶的功能,参与了虫休白噬：弓7侈虫SCD的缺失对速殖子阶段的生长繁殖没有显著影响。本研究初步探索了弓形虫SCD的生物学功能,为今后研究其作用机制捉供了科学依据。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasite, can be parasitic ten almost all warm blooded animal and human cells. Toxoplasma gondii in the parasitic life, out from the host intake pipe can be a variety of nutrients, but the body still has a relatively perfect metabolic system, can decompose and synthesis necessary. The ability of Toxoplasma gondii de novo synthesis of unsaturated fatty acids, but it is unclear whether Toxoplasma can synthesize unsaturated fatty acids. Therefore, we carried out the following research. Known stearoyl coenzyme A desaturase (stearoyl-coenzyme A, desaturase, SCD) is a key enzyme in the synthesis of unsaturated fatty acids the protein similarity comparison. Through the discovery, and Plasmodium SCD similar presumption of acyl coenzyme A of 54% desaturase gene in Toxoplasma gondii (ToxoDB No. TGGT1_238950). Bioinformatics analysis of the gene encoding. The protein has a typical desaturase domain. By reverse transcription PCR obtained 3129 full-length BP encoding sequence of the gene from Toxoplasma gondii RH strain, the sequence is completely consistent with TGGT1_238950. Then, the protein was truncated prokaryotic expression and preparation of mouse antibody, Western blotting results showed that the expression of the protein Toxoplasma gondii tachyzoites in the protein molecular weight is about 120 kDa, in line with expectations. At the same time to construct HA label endogenous marker strains of this gene by indirect immunofluorescence assay, found insect endogenous protein located in the endoplasmic reticulum, and other desaturase consistent positioning. Therefore, the presumption of Toxoplasma gondii stearoyl coenzyme A desaturase, namely Toxoplasma SCD (TgSCD). The cyclopropene fatty acids (e.g. sterculic acid) are inhibitors of SCD. We choose sterculic acid and its structure and several similar methyl in vitro anti Toxoplasma drugs Effect experiment, found the proliferation and release them to a different degree of inhibition of intracellular Toxoplasma gondii. Among them, sterculic acid inhibition of Toxoplasma Iabour minimum, but has obvious toxic effect on the host cells; another containing methoxy drugs (No. INT21) had significant inhibitory effects on Toxoplasma gondii and, at a high concentration of host cells without obvious toxicity. Thereafter, the experimental drug efficacy in mice, intraperitoneal injection of INT21 can make the Pru strain infected mice brain worm was significantly reduced, but no significant protective effect on mice infected by the RH strain. In order to further study the SCD in the development and proliferation of Toxoplasma gondii as it is, in the conditional Toxoplasma gondii strains' TATi with CRISPR/Cas9 technology in SCD knockout strains. By pyrethroid plaque assay and proliferation experiments showed that the pest invasion on development and reproduction, no significant changes to the phenotype; Rats inoculated with 1000 tachyzoites, SCD deletion strains and the virulence of different strains of mice TATi were not significant, suggesting that Toxoplasma gondii tachyzoite stage growth depends on SCD is not high, there may be other factors replace or compensate for its role in the absence of SCD. For further study on SCD in worms in function, constructed over expression of SCD strains of Toxoplasma gondii. Overexpression of fatty acid found detection of strains, the body of oleic acid increased in proportion to prove Toxoplasma SCD plays a desaturase function in the parasite strains in vitro. Overexpression phenotype observed that overexpression of SCD strain proliferation slowed down, invasion ability decreased; mice were inoculated with experiment, expression of virulence in mice than wild strain. The level of detection of Toxoplasma gondii SCD overexpression occurred in cases of stress by TgIF2 ER alpha phosphorylation and gene expression, and the history of easy autophagy and eight wide . in summary, this study makes clear the SCD of Toxoplasma gondii, and can play the function of desaturase, involved in insect Hugh autophagy: loss of 7 SCD of luxury arch insect tachyzoite stage had no significant effect on growth and reproduction. This study explore the biological function of Toxoplasma SCD, for further studies the mechanism and provide a scientific basis.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.7

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