日本血吸虫脱尾活童虫表膜结合肽的亲和筛选与功能鉴定

发布时间：2018-01-20 06:03

本文关键词：日本血吸虫噬菌体肽库差异淘选 MppZL4 日本血吸虫 MppZL4 靶向性拮抗性细胞毒性 RhB-ZL4 日本血吸虫脱尾童虫表膜结合肽 ZLW/pEGFP-C2质粒转染效率抗虫效果　出处：《中南大学》2011年博士论文　论文类型：学位论文

【摘要】：血吸虫病是一种重要危害人类身体健康和经济发展的寄生虫病,是世界主要公共卫生问题之一。当前,用于血吸虫病防治的方法,尽管有抗病原药物-吡喹酮用于人、畜化疗以控制病情和减少传染源,有杀中间宿主的氯硝柳胺等遥药用于人畜活动频繁的易感地带灭螺灭蚴,有丰富的家畜管理和人群健教的经验,但很难控制其流行传播,这说明现行防治技术尚存在不足。例如,在防治中起关键作用的吡喹酮,不仅只能在一个用药时点发挥作用,而且有研究发现其抗虫效果在降低。对此,本研究认为在抗感染疫苗和抗虫新药尚未问世之前,深入探索提高毗喹酮生物利用度和增强抗虫效果的方法是解决现场防治技术问题的重要课题之一。众所周知,杀成虫的吡喹酮和抗童虫的青篙素类药物用于人和家畜体内后表现代谢快和分布面大的特点,从而使药物的生物利用度低。如何来解决这一问题,本研究依据血吸虫生物学特性(需通过表膜吸收和结合宿主有关分子作为生长繁殖的因子)提出了虫体表膜有可能存在与外源分子发生特异性结合的分子,如能通过筛选获得,就有可能用其作靶向药物杀虫(提高药物利用度,减少药物所致毒副反应)。在本文中开展研究解决的关键问题是从血吸虫活虫体表膜中能筛选和鉴定出具有特异结合能力的多肽分子,并阐明其性质和功能,以期为进一步的靶向药物制剂等方面研究奠定工作基础。第一章日本血吸虫脱尾活童虫表膜结合噬菌体短肽的筛选与鉴定目的：利用噬菌体展示肽对日本血吸虫尾蚴脱尾活童虫作体外差异亲和淘选,以期获得对虫体具有拮抗或/和靶向作用的特异性短肽。方法：体外差异淘选与S.j脱尾活童虫结合、与尾蚴不结合的特异性短肽：将噬菌体12肽库用尾蚴预吸附后,取未与尾蚴结合的噬菌体肽库扩增后与脱尾童虫孵育,经洗脱、再扩增后,获取的目标肽库再次逆向吸附——扩增——淘选——扩增；经过3轮差异淘选,随机挑取15个克隆抽提DNA测序；通过氨基酸翻译和生物信息学分析测序结果；利用噬菌体回收实验、Western-blot及免疫组织化学检测分析所得噬菌体克隆与S.j童虫及成虫体被结合状态。结果：经3轮体外差异淘选获得4种不同序列的特异性M13噬菌体短肽MppZL6、MppZL4、MppZL1和MppZLO,其中MppZL0无插入片段；经生物信息学分析,MppZL6、MppZL4及MppZL1短肽均由8-9个亲水的极性氨基酸残基及疏水的非极性氨基酸残基交错构成,含精氨酸残基,等电点分别为6.07,8.75,8.50；BLAST分析表明,MppZL1与家鼠载脂蛋白B有53%(7/13)的同一性,MppZL6与Sj CHGC07116蛋白有69%(9/13)同一性,MppZL4与阴道毛滴虫自交第三代的表面抗原BspA样蛋白有80%(8/10)同一性。噬菌体回收实验显示克隆MppZL4与脱尾童虫的结合力显著高于MppZL6, MppZL1; Western blot结果显示,只有与MppZL4结合的成虫膜蛋白中有一条特异性条带,其大小为45.0kDa；免疫组化染色结果表明：MppZL4可与S.j毛蚴脱尾童虫、肺期童虫、肝期童虫及24天成虫结合。结论：通过噬菌体展示肽对活虫体作体外差异亲和淘选获得的特异性M13噬菌体MppZL4,可在小鼠体内特异性地与S.j的童虫和成虫表膜结合。第二章MppZL4及人工合成短肽ZL4的功能检测目的：通过MppZL4及人工合成的ZL4在体内外对日本血吸虫(S.j)的杀伤实验,检测ZL4的生物学功能,并从细胞水平上初步探讨ZL4的作用机制。方法：洗脱回收率检测MppZL4对S.j的靶向性：于小鼠感染S.j3d、及10d后,分别尾静脉注射MppZL41012pfu,15min后剖杀小鼠,心脏灌注,取出肝肺,洗脱噬菌体测定丰度,设立空白对照及阴性对照组；于小鼠感染S.j24d后,尾静脉注射MppZL41012pfu,15min后剖杀小鼠,心脏灌注冲虫,并取出肝肺,洗脱噬菌体测定丰度,同时洗脱虫体噬菌体并计数。体外杀伤实验检测MppZL4对S.j脱尾童虫的杀伤作用：无菌条件下MppZL4处理S.j脱尾童虫,每24小时美蓝染色后观察一次,连续观察72h,统计虫体死亡率,设置阳性对照、阴性对照及空白对照组。小鼠感染S.j24d后,尾静脉注射MppZL41012pfu,15min后剖杀小鼠取出虫体,无菌条件下制备S.j细胞,作免疫细胞化学观察MppZL4在细胞上的定位；体外培养MppZL4处理的S.j细胞1d后,MTT试剂盒检测其抑制率,并利用公式计算IC50。人工合成罗丹明标记的ZL4短肽RhB-ZL4,同法体外检测其对S.j童虫的结合功能及杀伤功能,体内检测其对S.j成虫的结合功能。结果：洗脱回收率检测MppZL4噬菌体靶向性结果显示：在感染小鼠体内,第3d时,肺组织MppZL4丰度为0.869±0.018,较高,而肝组织MppZL4丰度7.211±4.818,偏低；第10d则肺组织MppZL4丰度为0.239±0.065,较低,而肝组织MppZL4噬菌体丰度15.833±8.224,偏高；第24d时,各组噬菌体洗脱数无明显差异,但是虫体MppZL4洗脱丰度17.256±9.588远高于M13KE洗脱丰度0.202±0.056。体外杀伤实验观察到MppZL4致死率高于东方田鼠血清阳性对照组(P=0.000)：48h后,其死亡率已达到100%。免疫细胞化学检测结果显示,MppZL4主要沉积于S.j大圆细胞、三角形细胞及不规则细胞的细胞膜上。MTT检测细胞毒效应显示,MppZL4浓度为1010pfu时,即具有明显的细胞毒性(P=0.000),且随着MppZL4浓度的增高,毒性效应增强；IC50≈1021。 RhB-ZL4检测结果显示：RhB-ZL4体外与能与脱尾童虫体外特异性结合,其体外24hr及48hr致死率与MppZL4(?)(?)性对照组无显著性差异,72hr后死亡率为100%；体内能与S.j成虫特异性结合结论：MppZL4分子对血吸虫作用具有靶向性和一定的拮抗性及细胞毒性；RhB-ZL4可与日本血吸虫童虫及成虫特异性结合,对日本血吸虫童虫有一定的拮抗性。第三章ZLW/pEGFP-C2质粒的构建及其功能鉴定目的：观察日本血吸虫表膜特异结合肽合成基因构建的ZLW/pEGFP-C2质粒体外转染S.j脱尾童虫效率,了解其抗虫作用。方法：构建ZLW/pEGFP-C2质粒,运用二甲基亚砜(DMSO)作用下的高浓度质粒浸泡技术,将ZLW/pEGFP-C2对机械转化日本血吸虫童虫进行体外转染,以瞬时表达的EGFP为阳性标记,在倒置荧光镜下对虫体进行观察；转染后童虫培养48h,抽提虫体总RNA和全虫蛋白,分别用RT-PCR和Western blot检测ZLW基因和EGFP基因在童虫体内的表达；转染后虫体继续培养,于24、48、72及96h不同时点用美兰染色法鉴别虫体死活并计数；上述实验均设空质粒和正常童虫作平行对照。结果：成功构建ZLW/pEGFP-C2质粒,经ZLW/pEGFP-C2质粒转染的虫体在镜下观察显示有10%的转染率,其EGFP主要位于虫体皮层,以口、腹吸盘最为明显；RT-PCR扩增出259bp,片断大小与预期相符,经测序与ZLW序列一致；Western-blot证实EGFP基因在童虫虫体内有表达。抗虫效果显示,24及48h时,实验组、对照组死亡率无明显差异性(P24=0.600,P48=0.508)；72h后,实验组死亡率显著高于对照组(P72=0.000)；96h后实验组死亡率达到100%。结论：成功构建ZLW/pEGFP-C2质粒,DMSO作用下的高浓度质粒浸泡技术成功地将ZLW/pEGFP-C2质粒引入日本血吸虫童虫体内并获得表达；ZLW/pEGFP-C2质粒转染后有一定的抗童虫效果。
[Abstract]:Schistosomiasis is an important threat to human health and economic development of the parasitic disease, is one of the major public health problem in the world. At present, a method for schistosomiasis control, despite the anti drug praziquantel for people, and chemotherapy to control the disease and reduce the infection and kill the intermediate host of niclosamide and other drugs. For people in areas susceptible livestockactivities molluscacidal cercaricide, is rich in livestock management and population health education experience, but it is difficult to control the epidemic spread, this indicates that the current control technology is still inadequate. For example, play a key role in the prevention and treatment of praziquantel, not only can play a role in the use of a point. But studies have found that its effect in reducing the resistance. In this regard, this study suggests that before the anti infection vaccine and drug resistance has not yet come out, in-depth exploration of improving praziquantel bioavailability and enhance the anti insect effect. Law is one of the important topics to solve the problem of field control technology. As everyone knows, praziquantel killing schistosomula and adult resistance to artemisinin based drugs for fast metabolism and distribution characteristics and the performance of the animals, so that the bioavailability of the drug is low. How to solve this problem, on the basis of the the biological characteristics of Schistosoma japonicum (by membrane absorption and related molecules as factors with host growth and reproduction) proposed polypide surface membrane may have molecular binding specificity and exogenous molecules, such as through screening, it is possible to use the drug targeting insecticide (improving the bioavailability, reduce side reaction drug induced). To carry out research on key problems solved in this paper is from schistosome live to screening and identified peptides with specific binding ability of the parasite surface membrane, and clarify its nature and function, in order to It lays a foundation for further research on targeted drug preparation.
Screening and identification of membrane binding phage peptide of Schistosoma japonicum
Objective: to cercaria of Schistosoma japonicum schistosomula as body tail off live biopanning by phage display heterodyne different peptides, in order to get on the parasite with antagonistic or / and targeting specific short peptides.
Methods: in vitro panning and S.j difference tail off live schistosomula with specific peptide did not bind to measles: phage 12 mer peptide library with cercariae pre adsorption, phage display peptide library were not amplified and combined with cercariae and schistosomula incubated with tail, elution, amplification, target peptide library the adsorption was reverse again - after 3 rounds of panning, amplification; difference of panning, 15 clones from DNA sequencing; through the analysis of the sequencing results of amino acid translation and bioinformatics; using the phage recovery assay, phage cloning and S.j analysis of the schistosomula and adult body is combined with the state measured by Western-blot and immunohistochemistry..
Results: after 3 rounds of panning in vitro differences get 4 different kinds of sequence specific M13 phage peptides MppZL6, MppZL4, MppZL1 and MppZLO, of which MppZL0 insert; after analysis, bioinformatics, MppZL6, MppZL4 and MppZL1 peptide by nonpolar amino acid residues 8-9 a hydrophilic polar amino acid residues the hydrophobic and staggered form, containing arginine residues, the isoelectric point was 6.07,8.75,8.50; BLAST analysis showed that MppZL1 mice with apolipoprotein B 53% (7/13) of the same sex, MppZL6 69% and Sj CHGC07116 protein (9/13) with a MppZL4, and third generations of selfing of Trichomonas vaginalis BspA surface antigen like protein 80% (8/10). The same phage clone MppZL4 and recovery experiments indicate that the combination of tail off schistosomula was higher significantly than that in MppZL6, MppZL1; Western blot showed that only adult membrane protein and MppZL4 binding in a specific band, its size 45.0kDa; immunohistochemical staining results showed that MppZL4 and S.j into tail off schistosomula, lung stage schistosomula, hepatic schistosomula and 24 insect Tiancheng combination.
Conclusion: by the phage display peptide on live body as body of heterodyne biopanning obtained specific M13 phage MppZL4 in mice with specific S.j schistosomula and adult membrane binding.
Second chapter MppZL4 and functional detection of synthetic short peptide ZL4
Objective: to detect the biological function of ZL4 through the killing test of MppZL4 and synthetic ZL4 on Schistosoma japonicum (S.j) in vivo and in vitro, and to explore the mechanism of ZL4 from the cellular level.
Methods: the elution rate of S.j MppZL4 target detection to: in mice infected with S.j3d and 10d respectively after intravenous injection of MppZL41012pfu, 15min after the mice were sacrificed and heart perfusion, remove the liver and lung, eluted phage abundance determination, blank control and negative control group; in mice infected with S.j24d after intravenous injection MppZL41012pfu 15min, the mice were killed, at heart perfusion worms, and remove the liver and lung, determination of eluted phage abundance, eluted at worms bacteriophages and counting. Cytotoxicity assay of MppZL4 tail off schistosomula in vitro killing of S.j: MppZL4 S.j from the end of schistosomula sterile conditions, every 24 hours after methylene blue staining observation of a continuous observation of 72h, statistical insect mortality, set positive control, negative control and blank control group. Mice were infected with S.j24d after intravenous injection of MppZL41012pfu, 15min mice were killed to take out the body, under aseptic conditions. Preparation of S.j cells, for immunocytochemical MppZL4 in cells; S.j cells cultured in vitro after 1D MppZL4 treatment, MTT kit to detect the inhibitory rate of ZL4 peptide RhB-ZL4 and IC50. was calculated using synthetic Luo Danming labeled formula, with the combination of the S.j method in vitro detection of schistosomula and killing function the body function, to detect the S.j adult binding function.
Results: the detection rate of MppZL4 phage elution target to display results: in infected mice, the 3D, lung tissue MppZL4 abundance was 0.869 + 0.018, higher, and liver tissue MppZL4 abundance of 7.211 + 4.818, the 10d is low; lung tissue MppZL4 abundance was 0.239 + 0.065, lower, and MppZL4 of liver tissue phage abundance of 15.833 + 8.224, higher; at 24D, were eluted phage number have no obvious difference, but the insect abundance of 17.256 + 9.588 MppZL4 was much higher than that of M13KE was 0.202 + 0.056. abundance in vitro experiment observed the mortality of MppZL4 is higher than that of the East Tian serum positive control group (P=0.000) after:48h, the mortality rate has reached 100%. immunocytochemistry results showed that MppZL4 mainly deposited in large S.j cells, the cell membrane showed triangle cells and irregular cells on.MTT detection of cytotoxic effect, the concentration of MppZL4 is 1010pfu, which is Cell toxicity significantly (P=0.000), with the MppZL4 concentration increased, the toxic effect of enhancement; IC50 = 1021. RhB-ZL4. The results showed that: RhB-ZL4 in vitro and to tail off schistosomula in vitro and the specific binding of 24hr and 48hr in vitro, the mortality rate and MppZL4 (?) (?) there was no significant difference in control group. After 72hr, the mortality rate was 100%; the body can be combined with specific adult S.j
Conclusion: MppZL4 molecular targeted and antagonistic and cytotoxicity of schistosome; RhB-ZL4 and schistosomula and adult specific binding, there are certain antagonistic schistosomula. Construction and identification of the third chapter of ZLW/pEGFP-C2 plasmid
Objective: To observe the effect of Schistosoma japonicum membrane binding ZLW/pEGFP-C2 plasmid to construct two peptide synthesis genes with S.j tail off schistosomula efficiency, to understand the resistance effect.
Methods: ZLW/pEGFP-C2 plasmid was constructed, using two methyl sulfoxide (DMSO) plasmid immersion technology of high concentration under the action of ZLW/pEGFP-C2 on the mechanical transformation of schistosomula in vitro transfection, with transient expression of EGFP positive markers under inverted fluorescence microscope on the parasite were observed; 48h cultured schistosomula after transfection. Extraction of total RNA of Toxoplasma gondii and scorpion protein, respectively RT-PCR and Western blot detection of ZLW gene and EGFP gene expression in children in the insect body; after transfection worms cultured in 24,48,72 and 96h, the staining method for identifying insect death by methylene blue and counted; the experiment was set up in empty plasmid and normal schistosomula parallel control.
Results: the successful construction of ZLW/pEGFP-C2 plasmid, ZLW/pEGFP-C2 plasmid transfected by worms showed that the transfection rate of 10% was observed under the microscope, the main body is located in the EGFP cortex, with the mouth, abdominal sucker is most obvious; RT-PCR amplified 259bp fragment, in line with expectations, consistent with the ZLW sequencing confirmed that the EGFP gene Western-blot sequence; the expression in insect resistant effect in vivo. Children's show, 24 and 48h, the experimental group, the control group was no significant difference (P24=0.600, P48=0.508); after 72h, the experimental group was significantly higher than the control group (P72=0.000); experimental group after 96h mortality rate reached 100%.
Conclusion: the ZLW/pEGFP-C2 plasmid was successfully constructed, and the plasmid ZLW/pEGFP-C2 was successfully introduced into the Schistosoma japonicum larvae by DMSO under the action of high concentration plasmid immersion technology. The ZLW/pEGFP-C2 plasmid was transfected with certain plasmids.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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