细粒棘球蚴原头蚴包囊和原头蚴成虫抑制差减杂交文库中差异表达基因的筛选及分析

doi:10.11843/j.issn.0366-6964.2015.07.019

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (7): 1215-1223.doi: 10.11843/j.issn.0366-6964.2015.07.019

细粒棘球蚴原头蚴包囊和原头蚴成虫抑制差减杂交文库中差异表达基因的筛选及分析

赵莉^1#，石保新^1#，李军³，张壮志¹，马正海²，张旭¹，薛晶¹，金映红¹，张文宝^3*

（1.新疆畜牧科学院兽医研究所，乌鲁木齐 830000；2.新疆大学生命科学与技术学院重点实验室，乌鲁木齐 830046；3.新疆医科大学第一附属医院医学研究中心，新疆包虫病基础医学重点实验室，乌鲁木齐 830054）

收稿日期:2014-09-26 出版日期:2015-07-23 发布日期:2015-07-23
通讯作者: 张文宝，研究员，E-mail：wenbaozhang2013@163.com
作者简介:赵莉（1982-），女，江苏宿迁人，硕士，助理研究员，主要从事生物化学与分子生物学研究，E-mail：82285053@qq.com；石保新（1963-），男，河北馆陶人，研究员，E-mail:2423930978@qq.com。石保新与赵莉为同等贡献的第一作者
基金资助:
公益性行业（农业）科研专项项目（201103008）

The Screening and Analysis of Differentially Expressed Genes in Suppression Subtractive Hybridization Library with either Cyst (CW) or Adult Worm (AM) Development from Echinococcus granulosus Larval Protoscolex (PSC)

ZHAO Li^1# ，SHI Bao-xin^1# ，LI Jun³，ZHANG Zhuang-zhi¹，MA Zheng-hai²，ZHANG Xu¹，XUE Jing¹，JIN Ying-hong¹，ZHANG Wen-bao^3*

(1.Veterinary Research Institute of Animal Science Academy of Xinjiang Uygur Autonomous Region，Urumqi 830000，China；2.Xinjiang Key Laboratory of Biological Resources and Genetic Engineering，College of Life Science and Technology，Xinjiang University，Urumqi 830046，China；3.Xinjiang Hydatid Fundamental Medical Key Lab，First Teaching Hospital of Xinjiang Medical University，Urumqi 830054，China)

Received:2014-09-26 Online:2015-07-23 Published:2015-07-23

摘要/Abstract

摘要：

细粒棘球绦虫原头蚴（PSC）具有双向发育的特点，本文旨在利用SSH和生物信息学方法筛选原头蚴包囊（CW）和成虫（AW）特异性的基因。将PSC-CW和PSC-AW双相发育差减cDNA文库的差减PCR产物克隆入pGEM-T载体并测序，利用CAP3 sequence assembly在线软件、Blast2GO软件与GenBank数据库对测序结果进行分析，筛选原头蚴成囊和成虫特异性的基因，并探讨这些基因的功能。结果显示：对PSC-CW和PSC-AW文库进行扩增，分别得到280和200个阳性克隆，菌落PCR鉴定结果表明，这些克隆均插入200～1 000 bp片段。测序结果显示从PSC-CW和PSC-AW SSH文库中分别得到16和10个特异性基因。其中PSC-CW SSH文库中得到4个出现频率较高的基因，其余12个基因仅出现1次，其中5个为未知基因，已知基因分别编码细胞色素C氧化酶Ⅰ、热休克蛋白70和铁蛋白等功能蛋白质。PSC-AW SSH文库中得到10个基因，其中2个出现频率较高基因，8个基因仅出现1次，4个为未知基因，6个已知基因分别编码基质蛋白1、延伸因子1α和脂肪酸结合蛋白等功能蛋白质。本研究筛选获得原头蚴成囊和成虫发育时差异表达的基因，对这些基因的功能进行分析表明，PSC-CW SSH文库中多是与营养和能量转运功能的相关基因，而PSC-AW SSH文库中多为发育与分化相关的基因，这些基因的差异表达可能与原头蚴处于不同的生长环境和发育方向有关，同时也为棘球蚴病免疫诊断、药物筛选和疫苗研制提供候选基因。

Abstract:

According to the bidirectional development of the protoscolex (PSC) of Echinococcus granulosus，the study was conducted to screen the specifical genes of E.granulosus PSC development between a larval cyst and adult worm.The subtractive PCR products of the SSH library of E.granulosus PSC-adult (AW) and E.granulosus PSC-cyst (CW) were connected to the pGEM-T easy vector，and were cloned and sequenced.Totally，280 and 200 clones were obtained from PSC-CW and PSC-AM libraries，respectively，with the length of fragments 200 to 1000 bp.The clones were sequenced.Sequence alignment analysis showed that these sequences belong to 16 and 10 gene clusters representing 16 and 10 unique genes from PSC-CM and PSC-AW libraries respectively.As the genes are specifically or differentially expressed during the development of PSC to either adult worm or cyst，they are important for the parasite development and differentiation，thus can be candidates for drug and vaccine development.

中图分类号:

S852.734

赵莉，石保新，李军，张壮志，马正海，张旭，薛晶，金映红，张文宝. 细粒棘球蚴原头蚴包囊和原头蚴成虫抑制差减杂交文库中差异表达基因的筛选及分析[J]. 畜牧兽医学报, 2015, 46(7): 1215-1223.

ZHAO Li，SHI Bao-xin，LI Jun，ZHANG Zhuang-zhi，MA Zheng-hai，ZHANG Xu，XUE Jing，JIN Ying-hong，ZHANG Wen-bao. The Screening and Analysis of Differentially Expressed Genes in Suppression Subtractive Hybridization Library with either Cyst (CW) or Adult Worm (AM) Development from Echinococcus granulosus Larval Protoscolex (PSC)[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(7): 1215-1223.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2015.07.019

https://www.xmsyxb.com/CN/Y2015/V46/I7/1215

参考文献

［1］MCMANUS D P，ZHANG W，LI J，et al.Echinococcosis［J］.Lancet，2003，362(9392)：1295-1304.
［2］DIATCHENKO L，LAU Y F，CAMPBELL A P，et al.Suppression subtractive hybridization：a method for generating differentially regulated or tissue-specific cDNA probes and libraries［J］.Proc Natl Acad Sci U S A，1996，93(12)：6025-6030.
［3］GRUMOLATO L，ELKAHLOUN A G，GHZILI H，et al.Microarray and suppression subtractive hybridization analyses of gene expression in pheochromocytoma cells reveal pleiotropic effects of pituitary adenylate cyclase-activating polypeptide on cell proliferation，survival，and adhesion［J］.Endocrinology，2003，144(6)：2368-2379.
［4］REBRIKOV D V，BRITANOVA O V，GURSKAYA N G，et al.Mirror orientation selection (MOS)：a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization［J］.Nucleic Acids Res，2000，28(20)：E90.
［5］ERSFELD K，CRAIG P S.Cloning and immunological characterisation of Echinococcus granulosus ferritin［J］.Parasitol Res，1995，81(5)：382-387.
［6］SHONHAI A，BOSHOFF A，BLATCH G L.The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum［J］.Protein Sci，2007，16(9)：1803-1818.
［7］JENSEN A T，CURTIS J，MONTGOMERY J，et al.Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani(1)［J］.Biochim Biophys Acta，2001，1549(1)：73-87.
［8］JENSEN A T，ISMAIL A，GAAFAR A，et al.Humoral and cellular immune responses to glucose regulated protein 78- -a novel Leishmania donovani antigen［J］.Trop Med Int Health，2002，7(5)：471-476.
［9］IWAGAMI M，MONROY C，ROSAS M A，et al.A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador［J］.J Helminthol，2003，77(1)：33-38.
［10］MORGAN J A，DEJONG R J，KAZIBWE F，et al.A newly-identified lineage of Schistosoma［J］.Int J Parasitol，2003，33(9)：977-985.
［11］RAZO-MENDIVIL U J，LEÓN-RÈGAGNON V，PÉREZ-PONCE DE LEÓN G.Description of two new species of Glypthelmins Stafford，1905 (Digenea：Macroderoididae) in Rana spp.from Mexico，based on morphology and mtDNA and rDNA sequences［J］.Syst Parasitol，2004，59(3)：199-210.
［12］RAZO-MENDIVIL U，ROSAS-VALDEZ R，PÉREZ-PONCE DE LEÓN G.A new cryptogonimid (Digenea) from the Mayan cichlid，Cichlasoma urophthalmus (Osteichthyes：Cichlidae)，in several localities of the Yucatán Peninsula，Mexico ［J］.J Parasitol，2008，94(6)：1371-1378.
［13］LEÓN-RÈGAGNON V.Evidence of new species of Haematoloechus (Platyhelminthes：Digenea) using partial cox1 sequences［J］. Mitochondrial DNA，2010，21(Suppl 1)：12-17.
［14］LANE N.Biodiversity：On the origin of bar codes［J］.Nature，2009，462(7271)：272-274.
［15］OGEDENGBE J D，HANNER R H，BARTA J R.DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina，Apicomplexa，Alveolata)［J］.Int J Parasitol，2011，41(8)：843-850.
［16］KOEHLER C M，LEUENBERGER D，MERCHANT S，et al.Human deafness dystonia syndrome is a mitochondrial disease［J］.Proc Natl Acad Sci U S A， 1999，96(5)：2141-2146.
［17］CURRAN S P，LEUENBERGER D，SCHMIDT E，et al.The role of the Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins［J］.J Cell Biol，2002，158(6)：1017-1027.
［18］BLACKSTONE C，ROBERTS R G，SEEBURG D P，et al.Interaction of the deafness-dystonia protein DDP/TIMM8a with the signal transduction adaptor molecule STAM1［J］.Biochem Biophys Res Commun，2003，305(2)：345-352.
［19］BERGQUIST R，AL-SHERBINY M，BARAKAT R，et al.Blueprint for schistosomiasis vaccine development［J］.Acta Trop，2002，82(2)：183-192.
［20］CHUNCHOB S，GRAMS R，VIYANANT V，et al.Comparative analysis of two fatty acid binding proteins from Fasciola gigantica［J］. Parasitology，2010，137(12)：1805-1817.
［21］FONSECA C T，PACIFICO L G，BARSANTE M M，et al.Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone［J］.Microbes Infect，2006，8(9-10)：2509-2516.
［22］FAVRET C，VOEGTLIN D J.Speciation by host-switching in pinyon Cinara (Insecta：Hemiptera：Aphididae)［J］.Mol Phylogenet Evol，2004，32(1)：139-151.
［23］CHAVERRI P，BISCHOFF J F，EVANS H C，et al.Regiocrella，a new entomopathogenic genus with a pycnidial anamorph and its phylogenetic placement in the Clavicipitaceae［J］.Mycologia，2005，97(6)：1225-1237.
［24］XIAO N，QIU J，NAKAO M，et al.Echinococcus shiquicus n.sp.，a taeniid cestode from Tibetan fox and plateau pika in China［J］. Int J Parasitol，2005，35(6)：693-701.
［25］NAKAO M，MCMANUS D P，SCHANTZ P M，et al.A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes［J］.Parasitology，2007，134(Pt5)：713-722.
［26］HÜTTNER M，NAKAO M，WASSERMANN T，et al.Genetic characterization and phylogenetic position of Echinococcus felidis (Cestoda：Taeniidae) from the African lion［J］.Int J Parasitol，2008，38(7)：861-868.
［27］THOMPSON R C，MCMANUS D P.Towards a taxonomic revision of the genus Echinococcus［J］.Trends Parasitol，2002，18(10)：452-457.
［28］THOMPSON R C.The taxonomy，phylogeny and transmission of Echinococcus［J］.Exp Parasitol，2008，119(4)：439-446.
［29］SAARMA U，JGISALU I，MOKS E，et al.A novel phylogeny for the genus Echinococcus，based on nuclear data，challenges relationships based on mitochondrial evidence［J］.Parasitology，2009，136(3)：317-328.
［30］BADARACO J L，AYALA F J，BART J M，et al.Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus populations［J］.Exp Parasitol，2008，119(4)：453-459.
［31］HAAG K L，GOTTSTEIN B，AYALA F J.The EG95 antigen of Echinococcus spp.contains positively selected amino acids，which may influence host specificity and vaccine efficacy［J］.PLoS One，2009，4(4)：e5362.
［32］OKAMOTO M，NAKAO M，BLAIR D，et al.Evidence of hybridization between Taenia saginata and Taenia asiatica ［J］.Parasitol Int，2010，59(1)：70-74.
［33］NARAYANAN K，SRINIVAS R，RAMACHANDRAN A，et al.Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1 ［J］.Proc Natl Acad Sci U S A，2001，98(8)：4516-4521.

[1]	王正荣, 马勋, 张艳艳, 孙艳, 孟季蒙, 薄新文. 细粒棘球绦虫原头蚴、包囊壁和成虫circRNA差异表达分析[J]. 畜牧兽医学报, 2023, 54(8): 3474-3489.
[2]	杜小迪, 侯巍, 苏中华, 马青梅, 何雪, 华瑞其, 阳爱国, 杨光友. 细粒棘球绦虫泛素结合酶基因家族的生物信息学及表达分析[J]. 畜牧兽医学报, 2023, 54(6): 2605-2618.
[3]	李红, 李艳萍, 刘婷丽, 陈国梁, 王立群, 郭小腊, 骆学农. 多房棘球蚴感染对小鼠肝脂质代谢的影响[J]. 畜牧兽医学报, 2023, 54(1): 263-271.
[4]	李艳萍, 刘婷丽, 李红, 陈国梁, 王立群, 郭小腊, 骆学农. 多房棘球蚴诱导小鼠枯否氏细胞极化相关ceRNA调控网络的构建与分析[J]. 畜牧兽医学报, 2022, 53(12): 4410-4418.
[5]	王正荣, 马勋, 张艳艳, 孟季蒙, 薄新文. 基于转录组测序分析细粒棘球绦虫原头蚴与巨噬细胞RAW264.7免疫互作相关基因[J]. 畜牧兽医学报, 2022, 53(11): 3975-3988.
[6]	何雪, 王凝, 华瑞其, 杜小迪, 杨光友. 细粒棘球绦虫BAG3和EB1基因的克隆、表达及其在原头蚴细胞凋亡中的作用[J]. 畜牧兽医学报, 2022, 53(5): 1562-1575.
[7]	王正荣, 马勋, 张艳艳, 孟季蒙, 薄新文. Th1/Th2型免疫反应相关基因在巨噬细胞RAW264.7应对细粒棘球绦虫原头蚴刺激时的表达谱研究[J]. 畜牧兽医学报, 2022, 53(1): 250-262.
[8]	尚婧晔, 张光葭, 丹巴泽里, 王奇, 喻文杰, 何伟, 廖沙, 沈芸舟, 黄燕, 王谦, 钟波, 刘阳. 基于粪便样本的野生犬科动物物种鉴别与棘球绦虫感染调查[J]. 畜牧兽医学报, 2021, 52(12): 3546-3556.
[9]	潘耀谦, 孔令芸, 李鹏, 岳峰, 吴玉平, 刘兴友. 一种新型无钩豆状囊尾蚴的鉴定和18S DNA序列分析[J]. 畜牧兽医学报, 2019, 50(11): 2283-2289.
[10]	王正荣, 张艳艳, 马勋, 刘乙, 徐梦飞, 孟季蒙, 薄新文. 细粒棘球绦虫原头蚴lncRNA表达谱分析[J]. 畜牧兽医学报, 2019, 50(9): 1864-1873.
[11]	郭承, 万洁, 杨应东, 文建国, 刘俞辰, 古小彬, 谢跃, 杨光友. 脑多头蚴重组蛋白rTm16和rTm-GST的免疫保护效果分析[J]. 畜牧兽医学报, 2018, 49(12): 2707-2714.
[12]	王正荣, 薄新文, 张艳艳, 马勋, 卢平萍, 徐梦飞, 孟季蒙. 细粒棘球绦虫原头蚴microRNA表达谱分析[J]. 畜牧兽医学报, 2018, 49(11): 2477-2485.
[13]	徐梦飞, 卢平萍, 马勋, 张艳艳, 王炜烨, 孟季蒙, 王正荣, 薄新文. 细粒棘球绦虫蛋白质组学研究进展[J]. 畜牧兽医学报, 2018, 49(3): 466-476.
[14]	吴茂迪, 宋星桔, 闫敏, 阳爱国, 郭莉, 王凝, 谢跃, 古小彬, 杨光友. 细粒棘球绦虫半胱氨酸蛋白酶抑制剂基因的克隆、表达及诊断价值的初步评价[J]. 畜牧兽医学报, 2018, 49(3): 572-579.
[15]	黄兴, 徐璟, 王宇, 杨应东, 万洁, 郭承, 古小彬, 谢跃, 杨光友. 多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析[J]. 畜牧兽医学报, 2017, 48(12): 2374-2382.

细粒棘球蚴原头蚴包囊和原头蚴成虫抑制差减杂交文库中差异表达基因的筛选及分析

The Screening and Analysis of Differentially Expressed Genes in Suppression Subtractive Hybridization Library with either Cyst (CW) or Adult Worm (AM) Development from Echinococcus granulosus Larval Protoscolex (PSC)

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价