摘要
鸡球虫病是由艾美耳属的一种或几种球虫同时或先后寄生于鸡肠道黏膜上皮细胞所引起的疾病,造成的经济损失巨大。实践证明,抗球虫药物使用在鸡球虫的防治上具有重要作用。但抗药性问题限制了现有抗球虫药物的广泛应用,促使人们不得不寻找新的药物靶标。非甲羟戊酸(non-mevalonate MEP)类异戊二烯合成代谢(Isoprenoid biosynthesis)途径是广泛存在于植物、细菌和某些低等生物的一种重要合成代谢途径。已有的数据证明在疟原虫(Plasmodium spp)中存在MEP途径,并以此为药靶开发了膦铵霉素(fosmidomycin)这一新型抗疟药。本研究根据比较基因组学和生物信息学的原理和方法,预测出鸡球虫类异戊二烯合成代谢相关酶之一2-甲基赤藓糖醇-2,4-环焦磷酸合成酶(2C-Methyl-D-erythritol 2,4-cyclodiphosphate Synthase,EtMECS)的ORF序列,进行克隆、原核表达及亚细胞定位的研究。实验具体分为以下两个部分:
(1)EtMEC编码基因的生物信息学预测分析和基因克隆:分别以已报道的恶性疟原虫(P.falciparum)MECS的氨基酸序列(Gene Bank Acession Number AF279661)和刚地弓形虫(T.gondii)MECS的预测序列(55.m04628,http://www.toxodb.org)为参照,对Etenalla基因组数据库进行搜索比对,发现E.tenallaContig_0003528中含有MECS部分编码序列。以该序列编码的氨基酸序列对GeneBank数据库进行PSI-Blast分析预测EtMEC的ORF序列。以柔嫩艾美耳球虫第二代裂殖子总RNA为模板,用RT-PCR方法成功扩增出长261bp的EtMEC基因部分片段;在此基础之上,用RACE技术获得EtMEC基因的全长cDNA序列:测序结果表明,EtMEC全长cDNA共1625bp,5'和3'非编码区分别为27bp和380bp,EtMEC ORF长为1218bp;根据全长cDNA序列设计特异引物,成功扩增出包含整个ORF的序列片段。EtMEC的ORF编码一个由405个氨基酸组成的多肽,信号肽预测和多序列比对结果表明,其N-端包括一个由24个氨基酸组成的信号肽和128个氨基酸组成的转导肽,成熟的EtMEC蛋白共由253个氨基酸组成,分子量为37.6 KD。与其它物种MEC的氨基酸序列相似性在24-45%之间,进化树分析表明,EtMEC和TgMEC处于同一进化枝上。
(2)利用DNA重组技术,选原核表达载体pET-32a(+),利用E.coli Rosseta(DE3)重组表达EtMEC的成熟基因片段,结果重组载体能够得到表达;但pET-32a(+)-EtMEC重组表达蛋白大部分以包涵体的形式存在,采用不同的温度对表达条件进行优化,结果表明在37℃时重组蛋白的可溶性略高于16℃。选用37℃时纯化pET-32a(+)-EtMEC表达的重组蛋白,制备抗体,利用激光共聚焦技术研究EtMEC在E.tenella子孢子内的免疫荧光亚细胞定位。结果显示,EtMEC定位于E.tenella子孢子的顶质体,并发现部分子孢子可能具有多个顶质体。
Coccidiosis is an important avian disease which damages seriously to the poultry production.The strategies of controlling this important economically disease rely heavily on chemoprophylaxis.Due to increasing emergency of drug-resistant strains, exploring the new target for anti-coccidials had been eesiential.Non-mevalonate isoprenoid biosynthesis is an important anabolism pathway,which exsits generally in plant,bacterium and some primitive organism.The MEP pathway had proved to be exsited in plasmodium according to the data,and the new antimalarial,fosmidomycin, had been developed based on this pathway as the target.
In this paper,the ORF encoding the 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthetase of Eimeria tenella(EtMEC),one of the key enzymes in isoprenoid biosynthesis,was predicted by comparative genomics and bioinformatics.
There were two major experiments in this study:
(1) Bioinformatic predictive analysis and cloning of EtMEC.There was partial encoded MECS sequence in E.tenalla Contig_0003528 to be found by searching the E.tenalla genomic database with P.falciparum MECS amino acid array(Gene Bank Acession Number AF279661) and T.gondii MECS forecast arrays(55. m04628,http://www.toxodb.org) as references,respectively.The EtMEC ORF sequence was predicted with this encoed amino acid by carrying out PSI-BLAST. EtMEC partical ORF sequence was amplified by RT- PCR with total RNA extracted from E.tenella the second generation merozoites as template.The authentic 3'-and 5'-terminal sequences of EtMEC gene were obtained by using the rapid amplification of cDNA end(RACE) techniques.The full length of EtMEC gene is 1625bp,which contains the full ORF in length of 1218bp and includes 5'-untranslated region(5'-UTR) and a 3'-untranslated region (3'-UTR),which are 27bp and 380bp,respectively.A DNA fragment in length of 1625bp contained the full ORF was amplified by RT-PCR using a pair of special primers which was designed based on EtMEC cDNA sequence.The sequencing result indicated this sequence included a complete ORF with 1218 bp which encoded 405 amino acids.Amino acid sequences alignment of EtMEC against those of MEC from other model organisms by Clustal W showed 24-45%identity in amino acid composition.There is a leader peptide fragment in EtMEC involved in a signal peptide in length of 24 amino acids(initiated from N-terminal) and an apicoplast targeted transit peptide with 128 amino acids by Signal P3.0 prediction. This revealed the fragment of 759bp encoding the mature protein of EtMEC was composed of 253 amino acids with a predicted molecular weight of 37.6kD. Phylogenetic analysis of MECs indicated that EtMEC was in the same cluster with T.gondii MEC.Predicted conformation of mature EtMEC is similar to T.gondii MEC.
(2) The encoded region of EtMEC without N-terminal signal peptide was inserted into pET-32a(+) vector to construct the recombinant expresion vector,which was induced into E.coli Rosseta(DE3).The fusion protein was expressed successfully by inducing with IPTG.The result of SDS-PAGE indicated that the WM of the expressed protein was about 57.6 KD.Most of the recombinant protein was existed as the inclusion body.Using different temperature for optimizing condition,37℃is better than 16℃.The fusion protein was purified successfully by 6×His Resin column purification system.To determine the subcellar localization of EtMEC in Sporozoites,the EtMEC was located in the apicoplast of E.tenella by the immunofluorescenced visualization of confocal microscope.
引文
[1]Shirley,MW.A genetic blueprint for Emeria tenella.World poultry(Special issue on Coccidiosis),2003,13-14
[2]索勋,汪明,吴文学等.强效艾美耳牌鸡球虫苗Ⅰ型的田间实验[J].畜牧兽医学报,2001(23):265-269.
[3]孔繁瑶等.家畜寄生虫学[M](第二版).北京:中国农业大学出版社,1997
[4]Herz S.,Wungsintaweekul J.,Schuhr C.A.,et al.Biosynthesis of terpenoids YgbB protein converts 4-diphospho-cytidyl-2-c-methyl-D-erythritol 2-phosphate to 2C-methyl-D-eryth ritol-2.4-cyclodiphosphate[J].Proc.Natl.Acad.Sci.USA,2000,97:2 486-2 490.
[5]Rohdich F.,Wungsintaweekul J.,Feicht R.,et al.Cytidine 5-triphosphate-dependent biosynthesis of isoprenoids:Ygbp protein of E.coli catalyzes the formation of 4-diphocytidyl-2-C-methylerythritol[J].Proc.Natl.Acad.Sci.USA.1999,96:11758-11763.
[6]Rohdich F.,Wungsintaweekul J.,Hecht S.,et al.Biosynthesis of terpenoids:YchB protein of E.coli phosphoryates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol [J].Proc.Natl.Acad.Sci.USA.2000,97:12 062-12 067.
[7]Fichera M,Roose D S.1997.A plastid organelle as a drug target in apicomplexan parasite.[J].Nature.390:407-409
[8]Soldati D.1999.The apicoplast as a potential therapeutic target in T.gondii and other apicoplaxan parasite.[J].Parasitol.Today.15:5-7
[9]Altincicek B.,Kollas A.,Eberl M.,Wiesner J.,Sanderbrand S.,Hintz M.,et al.(2001)LytB,a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coil.[J].FEBS Lett.499:37-40
[10]Rohdich F.,Eisenreich W.,Wungsintaweekul J.,Hecht S.,Schuhr C.A.,Zenk M.H.et al.(2001)Biosynthesis of terpenoids 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase(IspF)from plasmodium falciparum.Eur.[J].Biochem.268:6302-6310
[11]Kishida H.,Wada T.,Unzai S.,Kuzuyama T.,Takagi M.,Terada T.et al.(2003) Structure and catalytic mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate(MECDP) synthase,An enzyme in the non-mevalonate pathway of isoprenoid synthesis.[J].Acta Cryst.D Biol.Cryst.59:23-31
[12]Ramos Valdivia AC.,Van der HR.,Verpoorte R.Isopentenyl diphosphate isomerase:a core enzyme in isoprenoid biosynthesis.A review of its biochemistry and function[J].Nat Prod Rep,1997,14:591
[13]Newman JD.,Chappell J.,Isoprenoid biosynthesis in plants:carbon partitioning within the cytoplasmic pathway[J].Crit Rev Biochem Mol Biol,1999,34:95
[14]Eisenreich W.,Schwarz M.,Cartayrade A.,et al.The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms[J].Chem Biol,1998,5:R221
[15]Lichtenthaler HK.,The 1-deoxy-d-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants[J].Annu Rev Plant Physiol Plant Mol Biol,1999,50:47
[16] Sprenger GA., Schorken U.,Wiegert T, et al. Identification of a thiamin-dependent synthase in E.coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isop- renoids, thiamin and pyridoxol [J].Proc.Natl.Acad.Sci.USA. 1997,91: 12857-12862.
[17] Sigerid G, Stephanieb M.,Hermanm S.,Isolation of the dxr gene of Zymomonas mobilis and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase[J]. FEM S Microbiology Letters, 2000, 191: 131-137.
[18] Soldati D.1999.The apicoplast as a potential therapeutic target in T.gondii and other apicoplaxan parasite. Parasitol.Today. 15:5-7
[19] Foth B.J.and McFadden G.I.2003,The apicoplast:a plastid in plasmodium falciparum and other apicomplexan parasites. Int. Rev. Cytol. 224:57-110
[20] Wilson RJM.2002.Progress with parasite plastids.J.Mol.Biol.319:257-274
[21] Foth BJ,Ralph SA,Tonkin CJ,et al.2003.Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science,299:705 -708
[22] Waller RF,Keeling PK,Donald PGK,et al.1998.Nuclear-encoded proteins target to the plastid in P. falciparum and T.gondii..PNAS.95:12352-12357
[23] Thomaen-Zieger N,Schachtner J,Seeber F.2003. Apicomplexan parasites contain a single lipoic acid synthase located in plastid.FEBS Letters.547:80-86
[24] Fichera M,Roose DS.1997.A plastid organelle as a drug target in apicomplexan parasite.Nature.390:407-409
[25] Waller RF,Reed MB,Cowman AF,et al. 2000.Protein trafficking to the plastid of Pfalciparum is via the secretory pathway.EMBO [J]. 19:1794-1782
[26] McFadden GL.,Waller RF.,Reith ME.,et al.1997.Plastid in parasites of humans.BioAssay,19: 1033-1040
[27] Crawford M J,Zhu G,Roose DS.2003.Both type I and type II fatty acid synthases in toxoplasma gondii. Molecular Parasitology Meeting XI V (2003) ,Abstract NO.14C
[28] Gardner MJ,Williamson DH,Wilson RJM.1991.A circular DNA in malaria parasites encodes an RNA polymerase like that of prolaryotrs and choroplasts.Mol.Biochem.Parasitol. 41: 115-124
[29] Zhu G,Marchewka MJ,Keithly JS.2000.Cryptosporidium parvum appears to lack a plastid genome Microbiology.146:315-321
[30] Cai X.M,Fuller L,Mcdougald LR,et al.2003.Apicoplast genome of coccodan Eimeria tenella.Gene,321:39-46
[31] Wilson RJM,Denny PW. 1996.Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum.J.Mol.Biol.261:155-172
[32] Roose DS,Crawford MJ,Donald RGK,et al.2002.Mining the plasmodium genome database to define organellar function:what does the apicoplast do? [J].Phil.Trans.Soc.Lond.357:35-46
[33]Sacchettini J.C.and Poulter C.D.1997.Creating isoprenoid diversity.[J].Science 277:178-179
[34]Rohmer M.,Seemann M,Horbach S.,Bringer-Meyer S.and Sahm H.1996.Glyeeraldehyde 3-phosphate and pvruvate as precursors of isoprenic units in an alternative pathway for terpenoid biosynthesis.[J].Am.Chem.Soc.118:2564-2566
[35]Lange BM.,Wildung MR.,McCaskill D.,et al..A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway[J].Proc Natl Acad Sci U S A,1998,95:2 100
[36]Lois L M.,Campos N.,Putra SR.,et al./Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate,a common precursor for isoprenoid,thiamin,and pyridoxol biosynthesis.[J].Proc Natl Acad Sci U S A,1998.95:2 105
[37]Eubanks L.M.and Poulter C.D.2003.Rhodobacter capsulatus 1 deoxy-D-xylulose 5-phosphate synthase:Steady-state kinetics and substrate binding.[J]Biochemistry 42:1140-1149
[38]Lois L.M,Rodriguez-Concepciom M,Gallego F.,Campos N.,2000.Carotenoid biosynthesis during tomato fruit development regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase.[J].Plant.22:503-513
[39]M(u∣¨)ller C.,Schwender J.,Zeidler J.and Lichtenthaler H.K.2000.Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis.[J].Biochem.Soc.Trans.28:792-793
[40]Coppens,Ⅰ.,Sinai,A.P.,Joiner,K.A.,2000.Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition.[J].Cell Biology 149,167-180.
[41]Takahashi S.,Kuzuyama T.,Watanabe H.,et al.A 12deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis[J].PNAS,1998,95:9 879
[42]Takahashi S.,Kuzuyama T.,Watanabe H.and Seto H.1998.A 1-deoxy-D-xylulose 5-phosphat reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an Alternative nonmevalonate pathway for terpenoid biosynthesis.[J].Proc.Natl.Acad.Sci.USA 95:9879-9884
[43]Koppisch A.T.,Fox D.T.,Blagg B.S.J.and Poulter C.D.2002.E.coli MEP synthase:steady-state kinetic analysis and substrate binding.[J].Biochemistry 41:236-243
[44]Jomaa H.,Wiesner J.,Sanderbrand S.,Altincicek B.,Weidemeyer C.,Hintz M.et al.1999Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.[J].Science 285:1573-1576
[45]Kuzuyama T.,Shimizu T.,Takahashi S.,and Seto H.1998.Fosmidomycin,a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis.[J].Tetrahedron Lett.39:7913-7916
[46]Fosmidomycin[3-(N-formyl-N-hydroxyamino)propyl-phosphonate]and FR-900098[3-(N-acetyl-N-hydroxy-amino)propylphosphonate]were synthesized by a combination of the protocols from work by Kamiya et al.and O" hler and Kanzler[T.Kamiya,M.Hashimoto,K.Hemmi,H.Takeno(Fujisawa Pharmaceutical Company),U.S.Patent 4,206,156 1980;E.O" hler and S.Kanzler,Synthesis 1995,539(1995)].Detailed protocols are available from the authors on request.
[47] Zeidler J., Schwender J., Muller C.,Wiesner J., Weidemeyer C, Beck E. et al.1998. Inhibition of the non-mevalonate l-deoxy-D-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. [J]. Z.Naturforsch. 53c:980-986
[48] Infected erythrocytes (200 ml per well,with 2%hematocrit and 0.4%parasitemia) were incubated with a serial dilution of the drugs for 48 hours. After the addition of 0.8-mCi [3H]-hypoxanthine in 30 ml of medium per well, the plates were further incubated for 24 hours. Incorporated radioactivity was counted after filtration on glass-fiber filters [M. L. Ancelin et al., Blood 91, 1.1998], Three P. falciparum strains were used for the sensitivity test (HB3, Honduras; A2,The Gambia; and Dd2, Indochina).
[49] The in vivo antimalarial activity was determined in P.vinckei infected mice with a modified 4-day suppressive test as described in work by Peters [W. Peters,Malaria, J. P. MECeier, Ed. (Academic Press, New York,1980), vol. 1, pp. 160 -161]. Mice were intraperitoneally inoculated with 10 infected erythrocytes on day 0. On day 1, successful infection was confirmed,and the treatment was started. The drugs were administrated every 8 hours, three times per day, with respect to their short plasma half-life time as previously published. [T. Murakawa H.,Sakamoto S.,Fukada T.,Konishi M.,Nishida.Antimicrob Agents Chemother. 21,224(1982)].
[50] Yajima S., Nonaka T., Kuzuyama T., Seto H. and Ohsawa K.2002.Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implifications of a flexible loop movement upon substrate binding. [J]. Biochem.131: 313-317
[51] Steinbacher S., Kaiser J., Eisenreich W., Huber R., Bacher A.and Rohdich F. 2003.Structural basis of fosmidomycin action revealed by the complex with IspC: implications for the catalytic mechanism and anti-malaria drug development.[J]..Biol. Chem. 278:18401-18407
[52] Kuzuyama T.,Takahashi S.,Takagi M.and Seto H. 2000. Characterization of 1-deoxy-D-xylulose 5-phosphate reductoismerase,an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residue. [J]. Biol. Chem. 275: 19928-19932
[53] Rohdich F, Wungsintaweekul J., Fellermeier M., Sagner S.,Herz S., Kis K. et al. 1999.Cytidine 5(?)-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol. [J].Proc. Natl. Acad. Sci. USA 96: 11758-11763
[54] Rohdich F., Wungsintaweekul I, Eisenreich W., Richter G.,Schuhr C. A., Hecht S. et al. 2000.Biosynthesis of terpenoids:4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana.[J].Proc. Natl. Acad. Sci. USA 97: 6451-6456
[55] Adam P., Eisenreich W., Fellermeier M., Hecht S., Rohdich F.,Schuhr C. A. et al.2000.The non-mevalonate isoprenoidpathway, [J].German Patent Application 10027821.3
[56] Luttgen H., Rohdich F., Herz S., Wungsintaweekul J., Hecht S., Schuhr C A. et al. 2000. Biosynthesis of terpenoids:YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol.[J].Proc.Natl..Acad.Sci.USA.97: 1062-1067
[57] Wungsintaweekul J.2001.Enzymes of the alternative terpenoid pathway in Escherichia coli, PhD Thesis, Technische Universitat Munchen, Germany
[58] Rohdic F, Wungsintaweekul J., Luttgen H., Fischer M.,Eisenreich W., Schuhr C. A. et al. 2000.Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato. [J].Proc. Natl. Acad. Sci. USA 97: 8251-8256
[59]Miallau L.,Alphey M.S.,Kemp L.E.,Leonard G A.,McSweeney S.M.,Hecht S.et al.2003.Biosynthesis of terpenoids:crystal structure of 4-diphosphocytidyl-2C-methyl-D-eryth-itol kinase,[J].Proc.Natl.Acad.Sci.USA 100:9173-9178
[60]Herz S.,Wungsintaweekul J.,Schuhr C.A.,Hecht S.,L(u∣¨)ttgen H.,Sagner S.et al.2000Biosynthesis of terpenoids:YgbB protein coverts 4-diphosphocytidyl-2C-methy-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate.[J].Proc.Natl.Acad.Sci.USA 97:2486-2490
[61]Rohdich F.,Eisenreich W.,Wungsintaweekul J.,Hecht S.,Schuhr C.A.,Zenk M.H.et al.2001.Biosynthesis of terpenoids.2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) from PlasmodiuM falciparum.Eur.[J].Biochem.268:3190-3197
[62]Seemann M.,Bui B.T.S.,Wolff M,Tritsch D.,Campos N.,Boronat A.et al.2002.Isoprenoid biosynthesis through the methylerythritol phosphate pathway:(E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase(GcpE) is a[4Fe-4S]protein.Angew.Chem.Int.Ed.41:4337-4339
[63]Vollmer,M.,Thomsen,N.,Wiek,S.and Seeber,F.2001.Apicomplexan parasites possess distinct nuclear-encoded,but apicoplast-localized,plant-type ferredoxin-NADP+ reductase and ferredoxin.[J].Biol.Chem.276,5483-5490.
[64]Hassan J.,Rene' C.R.,Nadine Englert.et al.2005.Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum [J].FEBS Letters.579;6433-6438
[65]Rohdich F.,Hecht S.,G(a∣¨)rtner K.,Adam P.,MECieger C.,Amslinger S.et al.2002.Studies on the nonmevalonate terpene biosynthetic pathway:metabolic role of IspH(LytB) protein.[J].Proc.Natl.Acad.Sci.USA 99:1158-1163
[66]Fellermeier M.,Raschke M.,Sagner S.,Wungsintaweekul J.,Schuhr C.A.,Hecht S.et al.2001.Studies on the nonmevalnate pathway of terpene biosynthesis.The role of 2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants.Eur.[J].Biochem.268:6302-6310
[67]Steinbacher S.,Kaiser J.,Wungsintaweekul J.,Hecht S.,Eisenreich W.,Gerhardt S.et al.2002.Structure of 2C-Methyl-D-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids.[J].Mol.Biol.316:79-88
[68]Huang Y S,Ge J,Zhang H M,et al.Purification and characterization of the Mycobacterium tuberculosis FabD2,a novel malonyl-CoA:AcpM transacylase of fatty acid synthase.[J].Protein Expr.Purif,2006,45393-45399
[69]BiMECam S G,Appels R,Oberholster A M.A workshop report on wheat genome sequencing:international genome research on wheat consortium.[J].Genetics,2004,168:1087-1096
[70]谢明权,吴惠贤,张键腓.Eimeria tenella裂殖子免疫原性的研究.畜牧兽医学报,1994,25(4):340-345
[71]J萨姆布鲁克D.W.拉塞尔著,黄培堂等译,分子克隆实验指南[M].(第三版).科学出版社.2002.8:522-525
[72]Frohman M A,Dush M K,Martin G R,et al.Rapid production of full-length cDNAs from rare transcripts:amplification using a single gene-specific oligonucleotide rimer.[J].PNAS,1988,85:8998-90
[73]钟涛,吴瑞英.cDNA末端快速扩增技术新进展.国外医学分子生物学分 册,2002,24(1):7-11
[74]李关荣,鲁成,夏庆友等.cDNA末端快速扩增技术(RACE)的优化与改良.生命科学研究,2003,7(3):189-197
[75]王少丽,盛承发,乔传令.cDNA末端快速扩增技术及其应用.遗传,2004,26(3):419-423
[76]索勋,李国清.鸡球虫病学[M].北京:中国农业出版社,1998
[77]Wakker RF,Raiph SA,Reed MB,et al.A type Ⅱ pathway fatty acid biosynthesis presents drug targets in Plasmodium falciparum.Antimicrob.Agents Chemother.2003.47:297-301
[78]倪灿荣,马大烈,戴益民主编.免疫组织化学实验技术及应用.北京:化学工业出版社.2006
[79]Cai X M,Fuller AL,McDougald LR,et al.Biochemical characterization of enoyl reductase involved in Type Ⅱ fatty acid synthesis in the intestinal coccidium Eimeria tenella (PhylumApicomplexa).FEMS[J].Microbiol Lett,2007,272:238-244
[80]Ferguson DJ,Campbell SA,Henriquez FL,et al.Enzymes of type Ⅱ fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.Int[J]Parasitol,2007,37:33-51