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
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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。
Section One:Differential Panning and Identify of peptides specifically binding to the tegument of live S.j schistosomula
Objective:The specific peptide with targeting and/or antagonism to the Schistosoma japonicum was gained by the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library.
Method:The specific peptides binding to schistosomula but cercarie were isolated by the techniqe of differential panning in vitro:The12peptide library without bingding to the cercarie was incubated and amplified with the schistosomula after force-absorbed with the cercarie, and the gained peptide library was eluted and amplified. After3times reverse absorption-amplification-panning-amplification, the DNA of15random phage clones was extract for sequencing. The sequences were analyzed through amino acids-translation and bioinformatics-analysis. The gained phage clones binding to schistosomulum and adult were detected and analyzed by the recovery rate of phage, Western-blot and immunohistochemistry.
Result:The four M13phage peptide clones with different sequences MppZL6, MppZL4, MppZL1and MppZLO, without the insertion element in MppZLO, were gained by3time's differential panning in vitro. Through bioinformatics-analysis, the peptide inserts from MppZL6, MppZL4and MppZL1, all containing arginine residue, were overlaped of8or9hydrophil polar amino acid residues and hydrophobe nonpolar amino acid residues, whose isoelectric point was6.07,8.75,8.50, respectively. BLAST analysis indicated that MppZL1showed53%(7/13) identities to apolipoprotein B of Mus musculus, MppZL6showed69%(9/13) identities to S.j CHGC07116protein and MppZL4showed80%(8/10) identities to surface antigen BspA-like influencing the functions of the hydrogenosome of T. v G3. The recovery rate of phage showed that the level of the phage clone MppZL4binding to schistosomulum was significantly higher than MppZL6and MppZL1. By Western blot, only the membrane protein of S.j adult binding to MppZL4clone displayed the specific band,45.0kDa. The results of immunohistochemistry suggested that ZL4could binding to Sj schistosomulum, lung-schistosomulum, hepatic-schistosomulum and adult worm.
Conclusion:The specific M13phage peptide MppZL4, using the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library, could specifically binded to the tegument of live Sj schistosomulum and adult worms in vitro of mouse.
Section Two:Functional testing of the MppZL4and RhB-ZL4
Objective:The biological functions of MppZL4and RhB-ZL4were detected by the killing-S.j-experiments in vivo and vitro, and the mechanism was preliminary study from the cellular level.
Method:Elution rate test the targeting of MppZL4binding to S.j:The mice, infected with S.j3rd day and10th day, were injected with MppZL41012pfu through tail vein, respectively, and were killed after15min. The phage MppZL4were eluted and counted from the lung and liver organs of the infected mice. At the same time, the control group and blank group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and killed after15min. The phage MppZL4were eluted and counted from the lung, liver organs and the worms of the infected mice. MppZL4phage lethal effect in vitro:S.j schistosomula were treated with MppZL4in axenic condition and observated every24h for72h through methylene blue stain, while the worms' mortality was counted. The positive control, negative control and blank control group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and sacrificed after15min. The S.j cells were manufactured used the worms from the mice in sterile condition and detected MppZL4by immunocytochemistry. The S.j cells were treated by MppZL4for one day whose inhibition rate was studied by MTT kit through IC50. The Rhodamine-labeled peptide RhB-ZL4were synthetic and detected the binding of S.j schistosomula in vitro and adult in vivo and the killing function of S.j schistosomula in vitro by the same ways.
Result:The results of elution rate showed, that the abundance of MppZL4in the lung tissue was0.869±0.018and the liver MppZL4abundance was7.211±4.818at the3rdday, whlie the abundance of MppZL4in the lung tissue was0.239±0.065lower than the3rd day and the liver MppZL4abundance was15.833±8.224at the10th day higher than the3rd day, but the abundance of phage eluted lung or liver tissue showed no significant difference among each group while the abundance of MppZL4eluted from worm was17.256±9.588and much higher than M13KE eluted from worm was0.202±0.056at the24th d. The death rate of MppZL4lethal effect in vitro is higher than fortis serum positive control group (P=0.000) and its mortality rate had reached100%after48hr. Immunocytochemical stain displaied that MppZL4phage mainly deposited the cell envelope of big round cells, triquetrous cells and irregular cells of Sj. MTT test illustrateed that MppZL41016pfu possessed significant cytotoxicity (P=0.000), and with the higher concentration of MppZL4, the cytotoxicity enhanced; IC50≈1021. The results showed:RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse. The death rate of MppZL4lethal effect in vitro showed no significant difference between the MppZL4positive control group and its mortality rate had reached100%after72h.
Conclusion:The specific MppZL4possess targeting and cytotoxicity for S.j. RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse, and possessed the cytotoxicity for S.j schistosomulum, too
Section Three:Construction and functional identification of ZLW/pEGFP-C2
Objective:To research the efficiency of ZLW/pEGFP-C2plasmid transfection into Schistosoma japonicum (Sj) schistosomula and observe the survival of the worms in vitro.
Method:After plasmids of ZLW/pEGFP-C2was constructed and transfected into Sj schistosomula, worms off the end of mechanical, using the immersion method containing0.75%DMSO and the high concentration of plasmid in vitro. The GFP as positive marker, the worm was observed under inverted fluorescence microscope; the presence of the transgenes ZLW and EGFP in Sj were confirmed by RT-PCR and western blot, respectively, with total RNA and proteins from transfect schistosomula and cultured for48hours. After transfect, the worms cultured24,48,72and96hours were respectively stained with methylene blue, observed determining and counted dead or live worm based on color availability. All experiments contained two parallel control groups of pEGFP-C2empty plasmid and TBS.
Result:The plasmid of ZLW/pEGFP-C2was constructed successfully. The transfect efficiency showed that the transfect rate was about10%and the fluorescence of EGFP was mainly localized in tegument of the worms especially predominate around the oral sucker and ventral sucker on the worms. The expected size of259bp fragment was successfully amplified by RT-PCR and its result of sequencing was consistent with the ZLW sequence, and the EGFP gene expression in worms was confirmed with western blot analysis. The results of affecting the worms to survive showed that the mortalities between experimental group and control groups showed no significant difference at24and48hours (P24=0.600, P48=0.508), but the mortality rate of experimental group was significantly higher than control groups at72hours (P72=0.000) and was100%after96h.
Conclusion:This study proved that ZLW/pEGFP-C2plasmid has been successfully introduced into the juvenile Schistosoma japonicum using the high concentration of plasmid immersion under0.75%DMSO in vitro and the survival of transfect parasites was enable certain effects.
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。
Section One:Differential Panning and Identify of peptides specifically binding to the tegument of live S.j schistosomula
Objective:The specific peptide with targeting and/or antagonism to the Schistosoma japonicum was gained by the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library.
Method:The specific peptides binding to schistosomula but cercarie were isolated by the techniqe of differential panning in vitro:The12peptide library without bingding to the cercarie was incubated and amplified with the schistosomula after force-absorbed with the cercarie, and the gained peptide library was eluted and amplified. After3times reverse absorption-amplification-panning-amplification, the DNA of15random phage clones was extract for sequencing. The sequences were analyzed through amino acids-translation and bioinformatics-analysis. The gained phage clones binding to schistosomulum and adult were detected and analyzed by the recovery rate of phage, Western-blot and immunohistochemistry.
Result:The four M13phage peptide clones with different sequences MppZL6, MppZL4, MppZL1and MppZLO, without the insertion element in MppZLO, were gained by3time's differential panning in vitro. Through bioinformatics-analysis, the peptide inserts from MppZL6, MppZL4and MppZL1, all containing arginine residue, were overlaped of8or9hydrophil polar amino acid residues and hydrophobe nonpolar amino acid residues, whose isoelectric point was6.07,8.75,8.50, respectively. BLAST analysis indicated that MppZL1showed53%(7/13) identities to apolipoprotein B of Mus musculus, MppZL6showed69%(9/13) identities to S.j CHGC07116protein and MppZL4showed80%(8/10) identities to surface antigen BspA-like influencing the functions of the hydrogenosome of T. v G3. The recovery rate of phage showed that the level of the phage clone MppZL4binding to schistosomulum was significantly higher than MppZL6and MppZL1. By Western blot, only the membrane protein of S.j adult binding to MppZL4clone displayed the specific band,45.0kDa. The results of immunohistochemistry suggested that ZL4could binding to Sj schistosomulum, lung-schistosomulum, hepatic-schistosomulum and adult worm.
Conclusion:The specific M13phage peptide MppZL4, using the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library, could specifically binded to the tegument of live Sj schistosomulum and adult worms in vitro of mouse.
Section Two:Functional testing of the MppZL4and RhB-ZL4
Objective:The biological functions of MppZL4and RhB-ZL4were detected by the killing-S.j-experiments in vivo and vitro, and the mechanism was preliminary study from the cellular level.
Method:Elution rate test the targeting of MppZL4binding to S.j:The mice, infected with S.j3rd day and10th day, were injected with MppZL41012pfu through tail vein, respectively, and were killed after15min. The phage MppZL4were eluted and counted from the lung and liver organs of the infected mice. At the same time, the control group and blank group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and killed after15min. The phage MppZL4were eluted and counted from the lung, liver organs and the worms of the infected mice. MppZL4phage lethal effect in vitro:S.j schistosomula were treated with MppZL4in axenic condition and observated every24h for72h through methylene blue stain, while the worms' mortality was counted. The positive control, negative control and blank control group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and sacrificed after15min. The S.j cells were manufactured used the worms from the mice in sterile condition and detected MppZL4by immunocytochemistry. The S.j cells were treated by MppZL4for one day whose inhibition rate was studied by MTT kit through IC50. The Rhodamine-labeled peptide RhB-ZL4were synthetic and detected the binding of S.j schistosomula in vitro and adult in vivo and the killing function of S.j schistosomula in vitro by the same ways.
Result:The results of elution rate showed, that the abundance of MppZL4in the lung tissue was0.869±0.018and the liver MppZL4abundance was7.211±4.818at the3rdday, whlie the abundance of MppZL4in the lung tissue was0.239±0.065lower than the3rd day and the liver MppZL4abundance was15.833±8.224at the10th day higher than the3rd day, but the abundance of phage eluted lung or liver tissue showed no significant difference among each group while the abundance of MppZL4eluted from worm was17.256±9.588and much higher than M13KE eluted from worm was0.202±0.056at the24th d. The death rate of MppZL4lethal effect in vitro is higher than fortis serum positive control group (P=0.000) and its mortality rate had reached100%after48hr. Immunocytochemical stain displaied that MppZL4phage mainly deposited the cell envelope of big round cells, triquetrous cells and irregular cells of Sj. MTT test illustrateed that MppZL41016pfu possessed significant cytotoxicity (P=0.000), and with the higher concentration of MppZL4, the cytotoxicity enhanced; IC50≈1021. The results showed:RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse. The death rate of MppZL4lethal effect in vitro showed no significant difference between the MppZL4positive control group and its mortality rate had reached100%after72h.
Conclusion:The specific MppZL4possess targeting and cytotoxicity for S.j. RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse, and possessed the cytotoxicity for S.j schistosomulum, too
Section Three:Construction and functional identification of ZLW/pEGFP-C2
Objective:To research the efficiency of ZLW/pEGFP-C2plasmid transfection into Schistosoma japonicum (Sj) schistosomula and observe the survival of the worms in vitro.
Method:After plasmids of ZLW/pEGFP-C2was constructed and transfected into Sj schistosomula, worms off the end of mechanical, using the immersion method containing0.75%DMSO and the high concentration of plasmid in vitro. The GFP as positive marker, the worm was observed under inverted fluorescence microscope; the presence of the transgenes ZLW and EGFP in Sj were confirmed by RT-PCR and western blot, respectively, with total RNA and proteins from transfect schistosomula and cultured for48hours. After transfect, the worms cultured24,48,72and96hours were respectively stained with methylene blue, observed determining and counted dead or live worm based on color availability. All experiments contained two parallel control groups of pEGFP-C2empty plasmid and TBS.
Result:The plasmid of ZLW/pEGFP-C2was constructed successfully. The transfect efficiency showed that the transfect rate was about10%and the fluorescence of EGFP was mainly localized in tegument of the worms especially predominate around the oral sucker and ventral sucker on the worms. The expected size of259bp fragment was successfully amplified by RT-PCR and its result of sequencing was consistent with the ZLW sequence, and the EGFP gene expression in worms was confirmed with western blot analysis. The results of affecting the worms to survive showed that the mortalities between experimental group and control groups showed no significant difference at24and48hours (P24=0.600, P48=0.508), but the mortality rate of experimental group was significantly higher than control groups at72hours (P72=0.000) and was100%after96h.
Conclusion:This study proved that ZLW/pEGFP-C2plasmid has been successfully introduced into the juvenile Schistosoma japonicum using the high concentration of plasmid immersion under0.75%DMSO in vitro and the survival of transfect parasites was enable certain effects.
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