本文關(guān)鍵詞： 日本血吸蟲 噬菌體 肽庫 差異淘選 MppZL4 日本血吸蟲 MppZL4 靶向性 拮抗性 細(xì)胞毒性 RhB-ZL4 日本血吸蟲 脫尾童蟲 表膜結(jié)合肽 ZLW/pEGFP-C2質(zhì)粒 轉(zhuǎn)染效率 抗蟲效果　出處：《中南大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：血吸蟲病是一種重要危害人類身體健康和經(jīng)濟(jì)發(fā)展的寄生蟲病,是世界主要公共衛(wèi)生問題之一。當(dāng)前,用于血吸蟲病防治的方法,盡管有抗病原藥物-吡喹酮用于人、畜化療以控制病情和減少傳染源,有殺中間宿主的氯硝柳胺等遙藥用于人畜活動頻繁的易感地帶滅螺滅蚴,有豐富的家畜管理和人群健教的經(jīng)驗(yàn),但很難控制其流行傳播,這說明現(xiàn)行防治技術(shù)尚存在不足。例如,在防治中起關(guān)鍵作用的吡喹酮,不僅只能在一個用藥時點(diǎn)發(fā)揮作用,而且有研究發(fā)現(xiàn)其抗蟲效果在降低。對此,本研究認(rèn)為在抗感染疫苗和抗蟲新藥尚未問世之前,深入探索提高毗喹酮生物利用度和增強(qiáng)抗蟲效果的方法是解決現(xiàn)場防治技術(shù)問題的重要課題之一。眾所周知,殺成蟲的吡喹酮和抗童蟲的青篙素類藥物用于人和家畜體內(nèi)后表現(xiàn)代謝快和分布面大的特點(diǎn),從而使藥物的生物利用度低。如何來解決這一問題,本研究依據(jù)血吸蟲生物學(xué)特性(需通過表膜吸收和結(jié)合宿主有關(guān)分子作為生長繁殖的因子)提出了蟲體表膜有可能存在與外源分子發(fā)生特異性結(jié)合的分子,如能通過篩選獲得,就有可能用其作靶向藥物殺蟲(提高藥物利用度,減少藥物所致毒副反應(yīng))。在本文中開展研究解決的關(guān)鍵問題是從血吸蟲活蟲體表膜中能篩選和鑒定出具有特異結(jié)合能力的多肽分子,并闡明其性質(zhì)和功能,以期為進(jìn)一步的靶向藥物制劑等方面研究奠定工作基礎(chǔ)。 第一章日本血吸蟲脫尾活童蟲表膜結(jié)合噬菌體短肽的篩選與鑒定 目的：利用噬菌體展示肽對日本血吸蟲尾蚴脫尾活童蟲作體外差異親和淘選,以期獲得對蟲體具有拮抗或/和靶向作用的特異性短肽。 方法：體外差異淘選與S.j脫尾活童蟲結(jié)合、與尾蚴不結(jié)合的特異性短肽：將噬菌體12肽庫用尾蚴預(yù)吸附后,取未與尾蚴結(jié)合的噬菌體肽庫擴(kuò)增后與脫尾童蟲孵育,經(jīng)洗脫、再擴(kuò)增后,獲取的目標(biāo)肽庫再次逆向吸附——擴(kuò)增——淘選——擴(kuò)增；經(jīng)過3輪差異淘選,隨機(jī)挑取15個克隆抽提DNA測序；通過氨基酸翻譯和生物信息學(xué)分析測序結(jié)果；利用噬菌體回收實(shí)驗(yàn)、Western-blot及免疫組織化學(xué)檢測分析所得噬菌體克隆與S.j童蟲及成蟲體被結(jié)合狀態(tài)。 結(jié)果：經(jīng)3輪體外差異淘選獲得4種不同序列的特異性M13噬菌體短肽MppZL6、MppZL4、MppZL1和MppZLO,其中MppZL0無插入片段；經(jīng)生物信息學(xué)分析,MppZL6、MppZL4及MppZL1短肽均由8-9個親水的極性氨基酸殘基及疏水的非極性氨基酸殘基交錯構(gòu)成,含精氨酸殘基,等電點(diǎn)分別為6.07,8.75,8.50；BLAST分析表明,MppZL1與家鼠載脂蛋白B有53%(7/13)的同一性,MppZL6與Sj CHGC07116蛋白有69%(9/13)同一性,MppZL4與陰道毛滴蟲自交第三代的表面抗原BspA樣蛋白有80%(8/10)同一性。噬菌體回收實(shí)驗(yàn)顯示克隆MppZL4與脫尾童蟲的結(jié)合力顯著高于MppZL6, MppZL1; Western blot結(jié)果顯示,只有與MppZL4結(jié)合的成蟲膜蛋白中有一條特異性條帶,其大小為45.0kDa；免疫組化染色結(jié)果表明：MppZL4可與S.j毛蚴脫尾童蟲、肺期童蟲、肝期童蟲及24天成蟲結(jié)合。 結(jié)論：通過噬菌體展示肽對活蟲體作體外差異親和淘選獲得的特異性M13噬菌體MppZL4,可在小鼠體內(nèi)特異性地與S.j的童蟲和成蟲表膜結(jié)合。 第二章MppZL4及人工合成短肽ZL4的功能檢測 目的：通過MppZL4及人工合成的ZL4在體內(nèi)外對日本血吸蟲(S.j)的殺傷實(shí)驗(yàn),檢測ZL4的生物學(xué)功能,并從細(xì)胞水平上初步探討ZL4的作用機(jī)制。 方法：洗脫回收率檢測MppZL4對S.j的靶向性：于小鼠感染S.j3d、及10d后,分別尾靜脈注射MppZL41012pfu,15min后剖殺小鼠,心臟灌注,取出肝肺,洗脫噬菌體測定豐度,設(shè)立空白對照及陰性對照組；于小鼠感染S.j24d后,尾靜脈注射MppZL41012pfu,15min后剖殺小鼠,心臟灌注沖蟲,并取出肝肺,洗脫噬菌體測定豐度,同時洗脫蟲體噬菌體并計(jì)數(shù)。體外殺傷實(shí)驗(yàn)檢測MppZL4對S.j脫尾童蟲的殺傷作用：無菌條件下MppZL4處理S.j脫尾童蟲,每24小時美藍(lán)染色后觀察一次,連續(xù)觀察72h,統(tǒng)計(jì)蟲體死亡率,設(shè)置陽性對照、陰性對照及空白對照組。小鼠感染S.j24d后,尾靜脈注射MppZL41012pfu,15min后剖殺小鼠取出蟲體,無菌條件下制備S.j細(xì)胞,作免疫細(xì)胞化學(xué)觀察MppZL4在細(xì)胞上的定位；體外培養(yǎng)MppZL4處理的S.j細(xì)胞1d后,MTT試劑盒檢測其抑制率,并利用公式計(jì)算IC50。人工合成羅丹明標(biāo)記的ZL4短肽RhB-ZL4,同法體外檢測其對S.j童蟲的結(jié)合功能及殺傷功能,體內(nèi)檢測其對S.j成蟲的結(jié)合功能。 結(jié)果：洗脫回收率檢測MppZL4噬菌體靶向性結(jié)果顯示：在感染小鼠體內(nèi),第3d時,肺組織MppZL4豐度為0.869±0.018,較高,而肝組織MppZL4豐度7.211±4.818,偏低；第10d則肺組織MppZL4豐度為0.239±0.065,較低,而肝組織MppZL4噬菌體豐度15.833±8.224,偏高；第24d時,各組噬菌體洗脫數(shù)無明顯差異,但是蟲體MppZL4洗脫豐度17.256±9.588遠(yuǎn)高于M13KE洗脫豐度0.202±0.056。體外殺傷實(shí)驗(yàn)觀察到MppZL4致死率高于東方田鼠血清陽性對照組(P=0.000)：48h后,其死亡率已達(dá)到100%。免疫細(xì)胞化學(xué)檢測結(jié)果顯示,MppZL4主要沉積于S.j大圓細(xì)胞、三角形細(xì)胞及不規(guī)則細(xì)胞的細(xì)胞膜上。MTT檢測細(xì)胞毒效應(yīng)顯示,MppZL4濃度為1010pfu時,即具有明顯的細(xì)胞毒性(P=0.000),且隨著MppZL4濃度的增高,毒性效應(yīng)增強(qiáng)；IC50≈1021。 RhB-ZL4檢測結(jié)果顯示：RhB-ZL4體外與能與脫尾童蟲體外特異性結(jié)合,其體外24hr及48hr致死率與MppZL4(?)(?)性對照組無顯著性差異,72hr后死亡率為100%；體內(nèi)能與S.j成蟲特異性結(jié)合 結(jié)論：MppZL4分子對血吸蟲作用具有靶向性和一定的拮抗性及細(xì)胞毒性；RhB-ZL4可與日本血吸蟲童蟲及成蟲特異性結(jié)合,對日本血吸蟲童蟲有一定的拮抗性。第三章ZLW/pEGFP-C2質(zhì)粒的構(gòu)建及其功能鑒定 目的：觀察日本血吸蟲表膜特異結(jié)合肽合成基因構(gòu)建的ZLW/pEGFP-C2質(zhì)粒體外轉(zhuǎn)染S.j脫尾童蟲效率,了解其抗蟲作用。 方法：構(gòu)建ZLW/pEGFP-C2質(zhì)粒,運(yùn)用二甲基亞砜(DMSO)作用下的高濃度質(zhì)粒浸泡技術(shù),將ZLW/pEGFP-C2對機(jī)械轉(zhuǎn)化日本血吸蟲童蟲進(jìn)行體外轉(zhuǎn)染,以瞬時表達(dá)的EGFP為陽性標(biāo)記,在倒置熒光鏡下對蟲體進(jìn)行觀察；轉(zhuǎn)染后童蟲培養(yǎng)48h,抽提蟲體總RNA和全蟲蛋白,分別用RT-PCR和Western blot檢測ZLW基因和EGFP基因在童蟲體內(nèi)的表達(dá)；轉(zhuǎn)染后蟲體繼續(xù)培養(yǎng),于24、48、72及96h不同時點(diǎn)用美蘭染色法鑒別蟲體死活并計(jì)數(shù)；上述實(shí)驗(yàn)均設(shè)空質(zhì)粒和正常童蟲作平行對照。 結(jié)果：成功構(gòu)建ZLW/pEGFP-C2質(zhì)粒,經(jīng)ZLW/pEGFP-C2質(zhì)粒轉(zhuǎn)染的蟲體在鏡下觀察顯示有10%的轉(zhuǎn)染率,其EGFP主要位于蟲體皮層,以口、腹吸盤最為明顯；RT-PCR擴(kuò)增出259bp,片斷大小與預(yù)期相符,經(jīng)測序與ZLW序列一致；Western-blot證實(shí)EGFP基因在童蟲蟲體內(nèi)有表達(dá)�？瓜x效果顯示,24及48h時,實(shí)驗(yàn)組、對照組死亡率無明顯差異性(P24=0.600,P48=0.508)；72h后,實(shí)驗(yàn)組死亡率顯著高于對照組(P72=0.000)；96h后實(shí)驗(yàn)組死亡率達(dá)到100%。 結(jié)論：成功構(gòu)建ZLW/pEGFP-C2質(zhì)粒,DMSO作用下的高濃度質(zhì)粒浸泡技術(shù)成功地將ZLW/pEGFP-C2質(zhì)粒引入日本血吸蟲童蟲體內(nèi)并獲得表達(dá)；ZLW/pEGFP-C2質(zhì)粒轉(zhuǎn)染后有一定的抗童蟲效果。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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