日本血吸虫抱雌沟蛋白相互作用因子的筛选及SjActin与SjTWF的研究
|
摘要
血吸虫病是一种在世界范围内广泛分布,由血吸虫感染引起的危害严重的人畜共患寄生虫病。血吸虫为吸虫中独特的雌雄异体形式,雌雄虫的合抱是血吸虫的生长发育的关键,抱雌沟蛋白是目前发现的唯一一种在雌雄虫合抱过程中进行传递的蛋白质。所以对抱雌沟蛋白及其相互作用蛋白的研究,对了解血吸虫生长发育的机理具有重要的意义
一、抱雌沟蛋白相互作用因子的筛选
利用本实验室构建的诱饵蛋白-血吸虫抱雌沟蛋白重组质粒pGBKT7-BD-SjGCP与成功构建的18日龄日本血吸虫酵母双杂交cDNA预转化文库进行抱雌沟蛋白相互作用因子的筛选。对筛选出的383个阳性文库质粒的外源插入片段进行序列测定得到131个外源插入序列,对其进行生物信息学分析,除去重复序列,共得到45个不同的有效基因序列。将得到的基因序列重新构建到pGADT7-AD载体上,与pGBK-BD-SjGCP进行回复杂交验证其相互作用,去除假阳性后得到了16个与血吸虫抱雌沟蛋白相互作用的蛋白的基因序列。本试验在筛选过程中采取了反复传代,并对阳性克隆进行回复杂交验证,保证了假阳性的去除,得到的16个蛋白经分析表明其有充分的与抱雌沟蛋白作用的可能性,但这些相互作用尚需进一步采取其他方式验证。
二、日本血吸虫Actin及Twinfilin蛋白的初步研究
为研究酵母双杂交所筛选出与SjGCP相互作用的SjACT和SjTWF蛋白,本文首先构建了这两种蛋白的重组表达载体,并在大肠杆菌中获得大量的原核表达产物。Westren-blot方法证实了表达出的蛋白具有良好的免疫原性,并且发现筛选出的SjActin蛋白为肌动蛋白家族中的Smooth Muscle Actin(SMA,Actin-α2)蛋白。利用抗SMA蛋白的单抗及SjGCP的多抗进行的激光共聚焦荧光共定位实验表明这两种蛋白在空间上都定位在血雌虫表膜上,从而证实了酵母双杂交筛选的可靠性。由Real-time方法分析SjActin和SjTWF在血吸虫发育不同时期的表达情况时发现SjActin和SjTWF的转录分别在血吸虫感染宿主后的23和21天较高。对SjTWF蛋白的免疫反应类型分析发现,免疫鼠脾细胞产生的TNF-α和IFN-γ量在SjTWF刺激后明显升高,差异极显著(P<0.001),而脾细胞产生的IL-10量在SjTWF刺激前后都比较高,之间没有显著差异(P>0.05),产生IL-4的量在SjTWF刺激后升高(P<0.05)。这说明SjTWF即刺激机体产生ThⅠ类型的免疫反应,也刺激机体产生ThⅡ类型的免疫反应。
三、RNA干扰日本血吸虫Actin, Twinfilin蛋白的研究
为更进一步确定SjACT和SjTWF在血吸虫生长发育过程中所起的作用,本文分别设计三对小RNA分子干扰体外培养血吸虫的SjACT和SjTWF蛋白表达。对干扰前后SjACT和SjTWF基因转录水平和蛋白表达水平的分析发现干扰获得成功,且发现干扰效果最佳的小分子干扰该蛋白的表达引起较高的血吸虫死亡率。对小RNA分子干扰对血吸虫形态影响的观察发现SjACT和SjTWF蛋白表达的下降都会影响血吸虫的运动,结果表明sjACT和sjTWF具有影响血吸虫运动和生长的作用。
Schistosomiasis is a serious parasitic disease infected by schistosome, which infects people and livestock and widely distributed in the world. Schistosome is a unique dioecious fluke, SGCP is the only protein which was found expressed especially at the gynaecophoral canal of male worms and transfer from male onto female, and SGCP was thought have some function on the development and mature of female worms. So the studies on SGCP and proteins interacted with SGCP would show some light on the mechanism of growth and development of schistosome.
一.Screening interactional factor of SjGCP protein
Recombinant plasmid pGBKT7-BD-SjGCP and 18d of Schistosoma japonicum cDNA library have been constructed ready for yeast Two-hybrid system. To screen of protein interact with SjGCP. , 131 inserted fragments was acquired from 383 positive recombinant plasmid, sequencing analysis of these fragments show that 45 different genes sequence we have got. Then these genes have been cloned into the plasmid of pGBKT7-BD-SjGCP to verify interaction of SjGCP protein by repeating Two-Hybrid, also Negative Control is done in order to remove false positive. Finally 16 genes sequence of the protein which interact with SjGCP were finally got. Because of using repeat subcultured yeast cell and repeat Two- Hybrid to remove false positive ,the results of 16 protein have be screened is reliable in this study .
二.SjACT and SjTWF protein preliminary research
In order to study of SjACT and SjTWF protein, screened by yeast Two-Hybrid. First, Recombinant plasmids of pET28a-SjACT and pET28a-SjTWF were constructed and SjACT and SjTWF proteins were expressed in E. coli. Good immunogencity was be verified using antiserum by western-bolt method, this results also confirmed that SjACT is a Smooth Muscle Actin (SMA,Actin-α2) of actin protein family. Then, sjACT and sjGCP protein was observed expression upper Schistosoma japonicum epidermis by using Laser Scanning Confocal Microscope, so this study shows reliability of interaction of SjGCP and SjACT protein. Additionally, Transcript level of SjACT and sjTWF is highest on 23d, 21d was detected out individually by using real-time PCR method. Finally we detected IL-10、TNF-α、IFN-γand IL-4 production following splenocyte activation with SjTWF protein,TNF-αand IFN-γproduction shows Significant differences from stimulated in relation to non-stimulated cells( P<0.001), but IL-10 production shows no significant differences (P>0.5), and IL-4 shows differences(P<0.5).This finding suggests a tendency of Th1 and Th2 type of immune response.
三. RNA interference SjACT and SjTWF protein
To determine the role of SjACT and SjTWF in Schistosoma japonicum growth and development, three siRNA was design to interference of SjACT and SjTWF protein expression on japonicum cultivated in vitro. analysising from transcriptional level and production of protein express, this experiment was succeed, and founding mortality of japonicum have positive correlation to effect of interference by siRNA. Observation form of Morphology, movement affected when interference of SjACT and SjTWF protein expression of japonicum. Conclusion that SjACT and SjTWF protein have some relationship to Schistosoma japonicum growth and development.
一、抱雌沟蛋白相互作用因子的筛选
利用本实验室构建的诱饵蛋白-血吸虫抱雌沟蛋白重组质粒pGBKT7-BD-SjGCP与成功构建的18日龄日本血吸虫酵母双杂交cDNA预转化文库进行抱雌沟蛋白相互作用因子的筛选。对筛选出的383个阳性文库质粒的外源插入片段进行序列测定得到131个外源插入序列,对其进行生物信息学分析,除去重复序列,共得到45个不同的有效基因序列。将得到的基因序列重新构建到pGADT7-AD载体上,与pGBK-BD-SjGCP进行回复杂交验证其相互作用,去除假阳性后得到了16个与血吸虫抱雌沟蛋白相互作用的蛋白的基因序列。本试验在筛选过程中采取了反复传代,并对阳性克隆进行回复杂交验证,保证了假阳性的去除,得到的16个蛋白经分析表明其有充分的与抱雌沟蛋白作用的可能性,但这些相互作用尚需进一步采取其他方式验证。
二、日本血吸虫Actin及Twinfilin蛋白的初步研究
为研究酵母双杂交所筛选出与SjGCP相互作用的SjACT和SjTWF蛋白,本文首先构建了这两种蛋白的重组表达载体,并在大肠杆菌中获得大量的原核表达产物。Westren-blot方法证实了表达出的蛋白具有良好的免疫原性,并且发现筛选出的SjActin蛋白为肌动蛋白家族中的Smooth Muscle Actin(SMA,Actin-α2)蛋白。利用抗SMA蛋白的单抗及SjGCP的多抗进行的激光共聚焦荧光共定位实验表明这两种蛋白在空间上都定位在血雌虫表膜上,从而证实了酵母双杂交筛选的可靠性。由Real-time方法分析SjActin和SjTWF在血吸虫发育不同时期的表达情况时发现SjActin和SjTWF的转录分别在血吸虫感染宿主后的23和21天较高。对SjTWF蛋白的免疫反应类型分析发现,免疫鼠脾细胞产生的TNF-α和IFN-γ量在SjTWF刺激后明显升高,差异极显著(P<0.001),而脾细胞产生的IL-10量在SjTWF刺激前后都比较高,之间没有显著差异(P>0.05),产生IL-4的量在SjTWF刺激后升高(P<0.05)。这说明SjTWF即刺激机体产生ThⅠ类型的免疫反应,也刺激机体产生ThⅡ类型的免疫反应。
三、RNA干扰日本血吸虫Actin, Twinfilin蛋白的研究
为更进一步确定SjACT和SjTWF在血吸虫生长发育过程中所起的作用,本文分别设计三对小RNA分子干扰体外培养血吸虫的SjACT和SjTWF蛋白表达。对干扰前后SjACT和SjTWF基因转录水平和蛋白表达水平的分析发现干扰获得成功,且发现干扰效果最佳的小分子干扰该蛋白的表达引起较高的血吸虫死亡率。对小RNA分子干扰对血吸虫形态影响的观察发现SjACT和SjTWF蛋白表达的下降都会影响血吸虫的运动,结果表明sjACT和sjTWF具有影响血吸虫运动和生长的作用。
Schistosomiasis is a serious parasitic disease infected by schistosome, which infects people and livestock and widely distributed in the world. Schistosome is a unique dioecious fluke, SGCP is the only protein which was found expressed especially at the gynaecophoral canal of male worms and transfer from male onto female, and SGCP was thought have some function on the development and mature of female worms. So the studies on SGCP and proteins interacted with SGCP would show some light on the mechanism of growth and development of schistosome.
一.Screening interactional factor of SjGCP protein
Recombinant plasmid pGBKT7-BD-SjGCP and 18d of Schistosoma japonicum cDNA library have been constructed ready for yeast Two-hybrid system. To screen of protein interact with SjGCP. , 131 inserted fragments was acquired from 383 positive recombinant plasmid, sequencing analysis of these fragments show that 45 different genes sequence we have got. Then these genes have been cloned into the plasmid of pGBKT7-BD-SjGCP to verify interaction of SjGCP protein by repeating Two-Hybrid, also Negative Control is done in order to remove false positive. Finally 16 genes sequence of the protein which interact with SjGCP were finally got. Because of using repeat subcultured yeast cell and repeat Two- Hybrid to remove false positive ,the results of 16 protein have be screened is reliable in this study .
二.SjACT and SjTWF protein preliminary research
In order to study of SjACT and SjTWF protein, screened by yeast Two-Hybrid. First, Recombinant plasmids of pET28a-SjACT and pET28a-SjTWF were constructed and SjACT and SjTWF proteins were expressed in E. coli. Good immunogencity was be verified using antiserum by western-bolt method, this results also confirmed that SjACT is a Smooth Muscle Actin (SMA,Actin-α2) of actin protein family. Then, sjACT and sjGCP protein was observed expression upper Schistosoma japonicum epidermis by using Laser Scanning Confocal Microscope, so this study shows reliability of interaction of SjGCP and SjACT protein. Additionally, Transcript level of SjACT and sjTWF is highest on 23d, 21d was detected out individually by using real-time PCR method. Finally we detected IL-10、TNF-α、IFN-γand IL-4 production following splenocyte activation with SjTWF protein,TNF-αand IFN-γproduction shows Significant differences from stimulated in relation to non-stimulated cells( P<0.001), but IL-10 production shows no significant differences (P>0.5), and IL-4 shows differences(P<0.5).This finding suggests a tendency of Th1 and Th2 type of immune response.
三. RNA interference SjACT and SjTWF protein
To determine the role of SjACT and SjTWF in Schistosoma japonicum growth and development, three siRNA was design to interference of SjACT and SjTWF protein expression on japonicum cultivated in vitro. analysising from transcriptional level and production of protein express, this experiment was succeed, and founding mortality of japonicum have positive correlation to effect of interference by siRNA. Observation form of Morphology, movement affected when interference of SjACT and SjTWF protein expression of japonicum. Conclusion that SjACT and SjTWF protein have some relationship to Schistosoma japonicum growth and development.
引文
1. Muschamp JW,Fong PP.Effects of the serotonin receptor ligand methio-thepin on reproductive behavior of the freshwater snail Biomphalaria glabrata:reduction of egglaying and induction of penileerection.J Exp Zool[J],2001,289(3):202-207.
2.郑江.我国血吸虫病防治的进展及面临的挑战.中国寄生虫学与寄生虫病杂志[J]2003,1(1):45-48
3. popie I,cioil D,Erasmus K A.Int.Prarasitiol[J],1984,12(4):166~185
4.臧炜,曹建平.血吸虫雌雄合抱及意义.国际医学寄生虫病杂技[J],2006,33(2):32-34
5.金亚美,林矫矫,程国锋,et al.日本血吸虫抱雌沟蛋白基因表达的性别与时相差异性研究.寄生虫与医学昆虫学报[J],2004(2):70-73
6.周述龙,林建银,蒋明森.血吸虫学(M) ,北京科学出版社, 2001.P96.
7. william s.aronstein,mette strand.A Glycoprotein antigen of schistosoma mansoni expressed on the gynecophoral canal of mature male worms.American society of tropical medicine and hygiene[J],1985,34(3):508-512
8. Osman Ahmed,Niles Edward G,Verjovski-Almeida Sergio,etal.Schistosoma mansini receptor II: role in host ligand-induced regulation of a schistosome target gene.PLOS pathogens[J],2006,2(6):0536-0550
9. Cheng GF,Lin JJ, Shi Y, et al.Dose-dependent inhibition of gynecophoral canal protein gene expression in vitro in the schistosome (Schistosoma japonicum) by RNA interference.Acta Biochim Biophys Sin[J],2005,37(6): 86-90
10. Fields S,Sternglanz R.The Two-hybrid shstem: an assay for protein-protein interactions.Traned Genet[J],1994,10:286-299
11. Guarente L.Strtegies for the identification of interacting protein.Proc Nat lacad Sci USA[J],1993,90:1639-1641
12.段素素,张云,王树惠等.酵母三杂交技术的应用策略及在病毒研究中的应用.国际病毒学杂志[J], 2006(2):37-41
13. U Putz,P Skehel and D Kuhl,A tri-hybrid system for the analysis and detection of RNA-protein interactions of outstanding interest. Nucleic Acids[J], 1996,24: 4838–4840
14. Licitra E J,Liu J O.A three-hybird system for detecting small ligand-protein receptor interactions.Proc Natl Acad Sci USA[J],1996,93(23):12817-12821
15. Ostrovsky O,Bengal E, Aronheim A.Induction of Terminal Differentiation by the c-Jun Dimerization Protein JDP2 in C2 Myoblasts and Rhabdomyosarcoma Cells.Biol Chem[J]. 2002,277(42): 40043-40054
16. Fire A,Xu S, Montgomery M,et al.Potent and specific genetic interference by double-stranded RNA inCaenorhabditis elegans.Nature[J],1998,391(6669):806-811
17. E Bernstein,S M Hammond,G J Hannon.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature[J],2010,404 (6775):293-296.
18. Tuschl T,Zamore PD,Sharp PA,et al.RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell[J], 2010,101(1):25-33
19. Hutvagner G,Mclachan J,Pasquinelli AE,et al.A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.Science[J],2001,293(5531):834-838
20. Caudy AA,Bernstein E,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature[J],2001,409(1818):363-366
21. Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and
22-nucleotide RNAs.Genes Dev[J],2003,17(4):438-442
22. DP Bartel.MicroRNAs: genomics, biogenesis, mechanism, and function.Cell[J],2004,116(2):281-297.
23. Skelly PJ,Da'dara A,Harn DA.Suppression of cathepsin B expression inSchistosoma mansoni by RNA interference.Parasitol[J],2003,33(4):363-369
24. Correnti JM,Brindley PJ,Pearce EJ.Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth. Mol Biochem Parasitol[J],2005, 143(2): 209-215
25. Nabhan JF,El-Shehabi F,Patocka N,et al.The 26S proteasome in Schistosoma mansoni: bioIFNormatics analysis,developmental expression,and RNA interference (RNAi) studies.Exp Parasitol[J],2007,117(3): 337-347
26. Osman A,Niles EG,Verjovski-Almeida S,et al.Schistosoma mansoni TGF-b receptor II: role in host ligand-induced regulation of a schistosome target gene.Plos Pathog[J],2006,2(6):54
27. Zhao ZR, Lei L, Liu M, et al. Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference.Exp Parasitol[J],2008, 119(3):379-384
28.贺淹才,肌动蛋白和肌动蛋白基因的研究进展.生命科学[J],2002,22(2):248 -249
29.王昭,周总光.肌动蛋白在炎症反应中的作用.中国微循环[J],2002,6(02): 17-31
30. Matsumoto Y,Perry G,Levine R,et al.Paramyosin and actin in Schistosomal teguments.Nature[J],1988,3(33):76
31. Davis A H,Blanton R,Clich P.Stage and sex specific difference in actin expression in Schistosoma mansoni.Molecular and Biochenical.Parasitology[J],1985, (17): 289
32.朱建国,林娇娇,冯新港,et al.日本血吸虫肌动蛋白cDNA的克隆表达及其免疫保护功能.中国人兽共患病杂志[J],2001,17(5):48-52
33.曲东明,韩梅,温进坤.肌动蛋白结合蛋白.细胞生物学杂志[J],2007,(29): 219-224
34. Thomas P.Stossel John Condeelis,Lynn Cooley,et al.Filamins as integrators ofcell mechanics and signaling.Nature[J],2001,2(2):138-144
35. Jie Lu,Gewei Lian, Robert Lenkinski,et al.Filamin B mutations cause chondrocyte defects in skeletal development.Human Molecular Genetics[J],2007(16):14 1661-1675
36. Mohamed MR,Shalaby KA, LoVerde PT,etal.Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin). Parasitology Research[J],2008,102 (5):1035-1042
1. LoVerd P.T,Chen L.Schistosoma female reproductive development.Parasitol Today [J],1991,7:303-308
2. Chen LL,Rkosh DM,LoVerde PT.Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene.Mol Parasitol[J],1991,52:53-59
3. LoVerde PT,Rekosh DM,Bobek LA,Developmentally regulated gene expression in Schistosoma.Exp Parasitol[J],1989,68(1): 116-120
4.周述龙,林建银,蒋明森. 2001.血吸虫学[M], (第2版) ,北京科学出版社P96.
5.刘珊珊,陈香梅,李蒙.BI-1作为酵母双杂交系统诱饵载体构建.国际遗传学杂志[J],2006,29(2):91-93
6. Li B,Fields S.Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybrid system.FASEB[J],1993,7 (10):957–963
7. Ye Q,Worman H J.Protein-protein interactions between human nuclear lamins expressed in yeast.Experimental Cell Res[J],1995,219(1):292–298.
8. Bartel P, Chien CT, Sternglanz R, et al. Elimination of false positives that arise in using the two hybrid system.BioTechniques[J],1993,14 (6):920 - 924
9. Pickart C M.Back to the future with ubiqutin.Cell[J],2004,116 (2):181-190
10. Turk B,Turk D,Turk V.Lysosomal cathepsins:structure,role in antigen processing and presentation,and cancer.Adv Enzyme Regul[M],2002,42:285-303
11.胡永轩,肖建华,曾桥,et al.日本血吸虫组织蛋白酶B内切酶基因结构和功能分析.中国人兽共患病杂志[J],2004,20(9):790-794
12. Cardoso FC,Paeffieo RN,Mortara PA,et a1.Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomie studies.Clin Exp Immunol[J],2006,144:382-391.
13. Fernanda C. Cardoso, Gilson C. Macedo,Elisandra Gava et al.Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection.PLoS Neglected Tropical Diseases[J],2008,2 (10):308-315
1.宇兴江,刘华杰,倪华.肌动蛋白解聚因子在动物生殖和胚胎发育中的作用.生理科学进展[M],2007,38(04):45-56
2. Guvakova M,Adams JC,Boettiger D.Functional role ofα-actinin,PI 3-kinase and MEK1/2 in insulin-like growth factorⅠreceptor kinase regulated motility of human breast cancer.cells[J],2002,(25)9:355-358
3. Goncharuk VN,Ross JS,Carlson JA.Actin-binding protein fascin expression in skin neoplasia.Journal of Cutaneous Pathology[J],2002,29(7):430-433
4. Fonseca CT,Brito CF,Alves JB,et al,IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-gamma and TNFalpha dependent pathway.Vaccine[J],2004,22: 503–510
5. Pac?′fico LG,Fonseca CT,Chiari L,Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental IFNection in a recombinant protein form but not as DNA vaccine.Immunobiology[J],2006,211: 97–104.
6. Muchaneta Kubara EC, Nahas AM. Myofibroblast phenotypes expression in experimental renal scarring. Nephrol Dial Transplant[J], 1997,12(5):904
7. william s,aronstein,mettestrand.A Glycoprotein antigen of schistosoma mansoni expressed on the gynecophoral canal of mature male worms.American society of tropical medicine and hygiene[J],1985,34(3):508-512
8.徐会娟,陈家旭,夏超明.血吸虫性肝纤维化形成及其免疫调节的分子机制.江苏大学学报[J],2003,13(5):466-468
9. Zhang L,Mi J,Yu Y,et al.IFN-γgene therapy by intrasplenic hepatocyte transplantation :a noval strategy for reversing hepatic fibrosis in Schistosoma japonicum IFNected mice . Parasite Immunol[J ],2001,23(1) :11– 17
10.翁红雷,蔡卫民,杨艳红.日本血吸虫感染兔I型和Ⅲ型胶原动态变化及干扰素-γ对其的作用.中国寄生虫学与寄生虫病杂志[J],2001,19 (1):26- 29
1. Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature[J],1998,391(6669) : 8062-8100.
2. Bosher JM,Lahouesse M.RNA interference : genetic wandand genetic watchdog.Nat Cell Biol [J ], 2000 , 2 (2) : E31.
3. Elbashir SM,Harbort h J,Lendeckel W,et al.Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature[J],2001,411 :494 - 498.
4.朱建国,林娇娇,冯新港,et al.日本血吸虫肌动蛋白cDNA的克隆表达及其免疫保护功能,中国人兽共患病杂志[J],2001,17(5):48-52
5. Mohamed MR,Shalaby KA,LoVerde PT,et al.Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin).Parasitology Research[J].2008,102 (5):1035-1042
6朱筱娟,曾宪录,宋朝霞.细胞核内肌动蛋白及其功能研究进展.科学通报[J]. 2004,49(11):1031-1034
7 Percipalle P,Zhao J,Pope B,et al.Actin bound to the heterogeneous nuclear ribonucleo protein hrp36 is associated with Balbiani ring mRNA from the gene to polysomes.Biol Chem[J],2001,153: 229~235
2.郑江.我国血吸虫病防治的进展及面临的挑战.中国寄生虫学与寄生虫病杂志[J]2003,1(1):45-48
3. popie I,cioil D,Erasmus K A.Int.Prarasitiol[J],1984,12(4):166~185
4.臧炜,曹建平.血吸虫雌雄合抱及意义.国际医学寄生虫病杂技[J],2006,33(2):32-34
5.金亚美,林矫矫,程国锋,et al.日本血吸虫抱雌沟蛋白基因表达的性别与时相差异性研究.寄生虫与医学昆虫学报[J],2004(2):70-73
6.周述龙,林建银,蒋明森.血吸虫学(M) ,北京科学出版社, 2001.P96.
7. william s.aronstein,mette strand.A Glycoprotein antigen of schistosoma mansoni expressed on the gynecophoral canal of mature male worms.American society of tropical medicine and hygiene[J],1985,34(3):508-512
8. Osman Ahmed,Niles Edward G,Verjovski-Almeida Sergio,etal.Schistosoma mansini receptor II: role in host ligand-induced regulation of a schistosome target gene.PLOS pathogens[J],2006,2(6):0536-0550
9. Cheng GF,Lin JJ, Shi Y, et al.Dose-dependent inhibition of gynecophoral canal protein gene expression in vitro in the schistosome (Schistosoma japonicum) by RNA interference.Acta Biochim Biophys Sin[J],2005,37(6): 86-90
10. Fields S,Sternglanz R.The Two-hybrid shstem: an assay for protein-protein interactions.Traned Genet[J],1994,10:286-299
11. Guarente L.Strtegies for the identification of interacting protein.Proc Nat lacad Sci USA[J],1993,90:1639-1641
12.段素素,张云,王树惠等.酵母三杂交技术的应用策略及在病毒研究中的应用.国际病毒学杂志[J], 2006(2):37-41
13. U Putz,P Skehel and D Kuhl,A tri-hybrid system for the analysis and detection of RNA-protein interactions of outstanding interest. Nucleic Acids[J], 1996,24: 4838–4840
14. Licitra E J,Liu J O.A three-hybird system for detecting small ligand-protein receptor interactions.Proc Natl Acad Sci USA[J],1996,93(23):12817-12821
15. Ostrovsky O,Bengal E, Aronheim A.Induction of Terminal Differentiation by the c-Jun Dimerization Protein JDP2 in C2 Myoblasts and Rhabdomyosarcoma Cells.Biol Chem[J]. 2002,277(42): 40043-40054
16. Fire A,Xu S, Montgomery M,et al.Potent and specific genetic interference by double-stranded RNA inCaenorhabditis elegans.Nature[J],1998,391(6669):806-811
17. E Bernstein,S M Hammond,G J Hannon.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature[J],2010,404 (6775):293-296.
18. Tuschl T,Zamore PD,Sharp PA,et al.RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell[J], 2010,101(1):25-33
19. Hutvagner G,Mclachan J,Pasquinelli AE,et al.A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.Science[J],2001,293(5531):834-838
20. Caudy AA,Bernstein E,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature[J],2001,409(1818):363-366
21. Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and
22-nucleotide RNAs.Genes Dev[J],2003,17(4):438-442
22. DP Bartel.MicroRNAs: genomics, biogenesis, mechanism, and function.Cell[J],2004,116(2):281-297.
23. Skelly PJ,Da'dara A,Harn DA.Suppression of cathepsin B expression inSchistosoma mansoni by RNA interference.Parasitol[J],2003,33(4):363-369
24. Correnti JM,Brindley PJ,Pearce EJ.Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth. Mol Biochem Parasitol[J],2005, 143(2): 209-215
25. Nabhan JF,El-Shehabi F,Patocka N,et al.The 26S proteasome in Schistosoma mansoni: bioIFNormatics analysis,developmental expression,and RNA interference (RNAi) studies.Exp Parasitol[J],2007,117(3): 337-347
26. Osman A,Niles EG,Verjovski-Almeida S,et al.Schistosoma mansoni TGF-b receptor II: role in host ligand-induced regulation of a schistosome target gene.Plos Pathog[J],2006,2(6):54
27. Zhao ZR, Lei L, Liu M, et al. Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference.Exp Parasitol[J],2008, 119(3):379-384
28.贺淹才,肌动蛋白和肌动蛋白基因的研究进展.生命科学[J],2002,22(2):248 -249
29.王昭,周总光.肌动蛋白在炎症反应中的作用.中国微循环[J],2002,6(02): 17-31
30. Matsumoto Y,Perry G,Levine R,et al.Paramyosin and actin in Schistosomal teguments.Nature[J],1988,3(33):76
31. Davis A H,Blanton R,Clich P.Stage and sex specific difference in actin expression in Schistosoma mansoni.Molecular and Biochenical.Parasitology[J],1985, (17): 289
32.朱建国,林娇娇,冯新港,et al.日本血吸虫肌动蛋白cDNA的克隆表达及其免疫保护功能.中国人兽共患病杂志[J],2001,17(5):48-52
33.曲东明,韩梅,温进坤.肌动蛋白结合蛋白.细胞生物学杂志[J],2007,(29): 219-224
34. Thomas P.Stossel John Condeelis,Lynn Cooley,et al.Filamins as integrators ofcell mechanics and signaling.Nature[J],2001,2(2):138-144
35. Jie Lu,Gewei Lian, Robert Lenkinski,et al.Filamin B mutations cause chondrocyte defects in skeletal development.Human Molecular Genetics[J],2007(16):14 1661-1675
36. Mohamed MR,Shalaby KA, LoVerde PT,etal.Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin). Parasitology Research[J],2008,102 (5):1035-1042
1. LoVerd P.T,Chen L.Schistosoma female reproductive development.Parasitol Today [J],1991,7:303-308
2. Chen LL,Rkosh DM,LoVerde PT.Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene.Mol Parasitol[J],1991,52:53-59
3. LoVerde PT,Rekosh DM,Bobek LA,Developmentally regulated gene expression in Schistosoma.Exp Parasitol[J],1989,68(1): 116-120
4.周述龙,林建银,蒋明森. 2001.血吸虫学[M], (第2版) ,北京科学出版社P96.
5.刘珊珊,陈香梅,李蒙.BI-1作为酵母双杂交系统诱饵载体构建.国际遗传学杂志[J],2006,29(2):91-93
6. Li B,Fields S.Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybrid system.FASEB[J],1993,7 (10):957–963
7. Ye Q,Worman H J.Protein-protein interactions between human nuclear lamins expressed in yeast.Experimental Cell Res[J],1995,219(1):292–298.
8. Bartel P, Chien CT, Sternglanz R, et al. Elimination of false positives that arise in using the two hybrid system.BioTechniques[J],1993,14 (6):920 - 924
9. Pickart C M.Back to the future with ubiqutin.Cell[J],2004,116 (2):181-190
10. Turk B,Turk D,Turk V.Lysosomal cathepsins:structure,role in antigen processing and presentation,and cancer.Adv Enzyme Regul[M],2002,42:285-303
11.胡永轩,肖建华,曾桥,et al.日本血吸虫组织蛋白酶B内切酶基因结构和功能分析.中国人兽共患病杂志[J],2004,20(9):790-794
12. Cardoso FC,Paeffieo RN,Mortara PA,et a1.Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomie studies.Clin Exp Immunol[J],2006,144:382-391.
13. Fernanda C. Cardoso, Gilson C. Macedo,Elisandra Gava et al.Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection.PLoS Neglected Tropical Diseases[J],2008,2 (10):308-315
1.宇兴江,刘华杰,倪华.肌动蛋白解聚因子在动物生殖和胚胎发育中的作用.生理科学进展[M],2007,38(04):45-56
2. Guvakova M,Adams JC,Boettiger D.Functional role ofα-actinin,PI 3-kinase and MEK1/2 in insulin-like growth factorⅠreceptor kinase regulated motility of human breast cancer.cells[J],2002,(25)9:355-358
3. Goncharuk VN,Ross JS,Carlson JA.Actin-binding protein fascin expression in skin neoplasia.Journal of Cutaneous Pathology[J],2002,29(7):430-433
4. Fonseca CT,Brito CF,Alves JB,et al,IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-gamma and TNFalpha dependent pathway.Vaccine[J],2004,22: 503–510
5. Pac?′fico LG,Fonseca CT,Chiari L,Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental IFNection in a recombinant protein form but not as DNA vaccine.Immunobiology[J],2006,211: 97–104.
6. Muchaneta Kubara EC, Nahas AM. Myofibroblast phenotypes expression in experimental renal scarring. Nephrol Dial Transplant[J], 1997,12(5):904
7. william s,aronstein,mettestrand.A Glycoprotein antigen of schistosoma mansoni expressed on the gynecophoral canal of mature male worms.American society of tropical medicine and hygiene[J],1985,34(3):508-512
8.徐会娟,陈家旭,夏超明.血吸虫性肝纤维化形成及其免疫调节的分子机制.江苏大学学报[J],2003,13(5):466-468
9. Zhang L,Mi J,Yu Y,et al.IFN-γgene therapy by intrasplenic hepatocyte transplantation :a noval strategy for reversing hepatic fibrosis in Schistosoma japonicum IFNected mice . Parasite Immunol[J ],2001,23(1) :11– 17
10.翁红雷,蔡卫民,杨艳红.日本血吸虫感染兔I型和Ⅲ型胶原动态变化及干扰素-γ对其的作用.中国寄生虫学与寄生虫病杂志[J],2001,19 (1):26- 29
1. Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature[J],1998,391(6669) : 8062-8100.
2. Bosher JM,Lahouesse M.RNA interference : genetic wandand genetic watchdog.Nat Cell Biol [J ], 2000 , 2 (2) : E31.
3. Elbashir SM,Harbort h J,Lendeckel W,et al.Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature[J],2001,411 :494 - 498.
4.朱建国,林娇娇,冯新港,et al.日本血吸虫肌动蛋白cDNA的克隆表达及其免疫保护功能,中国人兽共患病杂志[J],2001,17(5):48-52
5. Mohamed MR,Shalaby KA,LoVerde PT,et al.Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin).Parasitology Research[J].2008,102 (5):1035-1042
6朱筱娟,曾宪录,宋朝霞.细胞核内肌动蛋白及其功能研究进展.科学通报[J]. 2004,49(11):1031-1034
7 Percipalle P,Zhao J,Pope B,et al.Actin bound to the heterogeneous nuclear ribonucleo protein hrp36 is associated with Balbiani ring mRNA from the gene to polysomes.Biol Chem[J],2001,153: 229~235