堆型艾美耳球虫ATP酶基因的生物信息学分析及原核表达
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摘要
为研究堆型艾美耳球虫阳离子转运ATP酶(Eimeria acervulina Na+-K+-ATPase,Ea NKA)蛋白的生物学功能,根据Ea NKA基因(GenBank登录号:EU590120.1)设计1对特异性引物,以堆型艾美耳球虫孢子化卵囊cDNA文库为模板,对Ea NKA基因进行PCR扩增,将PCR扩增产物克隆入pUCM-T载体并进行测序鉴定,将测序获得的Ea NKA基因编码蛋白进行生物信息学分析。Ea NKA基因全长1157 bp,位于第81-368 bp之间,编码236个氨基酸,分子量约为25 kDa。编码蛋白主要位于内质网,不具有信号肽,编码蛋白主要位于内质网,不具有信号肽,有12个结构功能域,2个跨膜区,位于多肽N端的7个抗原表位。Blast分析显示其编码蛋白与堆型艾美耳球虫阳离子转运ATP酶相关蛋白序列(XP013248919、ACB97673)的同源性为100%。构建重组质粒p ET32a(+)-Ea NKA并进行测序鉴定,然后将重组质粒转化到大肠杆菌BL21感受态细胞,进行SDS-PAGE和Western blot表达鉴定。结果显示,构建的重组质粒可在原核细胞中成功表达45 kDa的融合蛋白。本研究成功获得Ea NKA基因的全长序列,并在原核细胞中成功表达,为今后研究该基因的功能及筛选基因工程疫苗候选分子奠定基础。
In order to study the biological function of Na+-K+-ATPase protein of Eimeria acervulina,(Eimeria acervulina Na+-K+-ATPase, Ea NKA), one pair of specific primers were designed according to the Ea NKA gene sequence deposited in GenBank(accession No. EU590120.1). The ORF of Ea NKA gene was amplified by PCR using the cDNA library of E. acervulina sporulated oocysts as a template. After sequence analysis, the structures and biochemical properties of Ea NKA protein were analyzed using bioinformatics software. The PCR fragment was ligated to the prokaryotic expression vector pET32 a(+) to construct the recombinant plasmid pET32 a(+)-Ea NKA. Then, the recombinant plasmid was transfected into BL21 cells for induction with IPTG. The results showed that the fulllength of Ea NKA was 1157 bp in length and ORF located between 81-788 bp, encoding 236 amino acids with a molecular weight of about25 kDa. The encoded protein mainly located in the endoplasmic reticulum and consisted of 12 structural domains, two transmembrane regions without a signal peptide. In addition, seven epitopes mainly located in the N-terminal polypeptide. Blast analysis showed that the encoded protein shared 100% sequence identity with ATPase-related protein of Eimeria acervulina sequence(XP013248919, ACB97673).Western blotting showed that a protein with molecular weight of 45 kDa in the transfected cells was strongly recognized by rabbit antiserum against E.acervulina sporulated oocysts. These results indicated that the full-length sequence of Ea NKA gene was obtained and expressed in prokaryotic cells, which laid the foundation for the screening of genetically engineered vaccine candidate molecules.
In order to study the biological function of Na+-K+-ATPase protein of Eimeria acervulina,(Eimeria acervulina Na+-K+-ATPase, Ea NKA), one pair of specific primers were designed according to the Ea NKA gene sequence deposited in GenBank(accession No. EU590120.1). The ORF of Ea NKA gene was amplified by PCR using the cDNA library of E. acervulina sporulated oocysts as a template. After sequence analysis, the structures and biochemical properties of Ea NKA protein were analyzed using bioinformatics software. The PCR fragment was ligated to the prokaryotic expression vector pET32 a(+) to construct the recombinant plasmid pET32 a(+)-Ea NKA. Then, the recombinant plasmid was transfected into BL21 cells for induction with IPTG. The results showed that the fulllength of Ea NKA was 1157 bp in length and ORF located between 81-788 bp, encoding 236 amino acids with a molecular weight of about25 kDa. The encoded protein mainly located in the endoplasmic reticulum and consisted of 12 structural domains, two transmembrane regions without a signal peptide. In addition, seven epitopes mainly located in the N-terminal polypeptide. Blast analysis showed that the encoded protein shared 100% sequence identity with ATPase-related protein of Eimeria acervulina sequence(XP013248919, ACB97673).Western blotting showed that a protein with molecular weight of 45 kDa in the transfected cells was strongly recognized by rabbit antiserum against E.acervulina sporulated oocysts. These results indicated that the full-length sequence of Ea NKA gene was obtained and expressed in prokaryotic cells, which laid the foundation for the screening of genetically engineered vaccine candidate molecules.
引文
[1]Blake D P,Alias H,Billington K J,et al.EmaxDB:availability of a first draft genome sequence for the apicomplexan Eimeria maxima[J].Mol Biochem Parasitol,2012,184(1):48-51.
[2]Burt S A,Tersteeg-zijderveld M H,JongeriusGortemaker B G,et al.In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals[J].Vet Parasitol,2013,191(3-4):374-378.
[3]Del Cacho E,Gallego M,Lee S H,et al.Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells(DC)and DC-derived exosomes[J].Vaccine,2011,29(21):3818-3825.
[4]Sun H,Wang L,Wang T,et al.Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis[J].Vaccine,2014,32(16):1869-1876.
[5]Gurunathan S,Wu C Y,Freidag B L,et al.DNAvaccines:a key for inducing long-term cellular immunity[J].Curr Opin Immunol,2000,12(4):442-447.
[6]刘晶,陈复辉,陈淑芳,等.钠钾ATP酶的临床研究进展[J].现代医学,2016,44(6):914-917.
[7]孙董董,林春花,李博勋,等.3种炭疽菌P型ATP酶基因家族分析[J].热带作物学报,2015,36(4):737-745.
[8]盛江筠,朱敏,黄正蔚,等.耐氟菌株及其亲代菌株ATP酶基因的DNA测序[J].上海第二医科大学学报,2005,25(4):340-341.
[9]王竹青,任宪云,高保全,等.三疣梭子蟹F型ATP酶β亚基(F-ATPaseβ)基因的克隆、组织表达及在家系近交中的变化[J].渔业科学进展,2018,39(1):97-106.
[10]马欢.小地老虎V-ATP酶A、B亚基的克隆及B亚基昆虫杆状病毒沉默载体构建的研究[D].杨凌:西北农林科技大学,2015.
[11]陈军营,岳润清,陈新建,等.小麦质膜质子ATP酶基因和启动子序列(登录号:AY829002)[J].分子植物育种,2005,3(6):903-904.
[12]张艳芳,孙瑞芬,郭树春,等.向日葵V-ATPase a3亚基基因的克隆及表达分析[J].中国生物工程杂志,2017,37(5):19-27.
[13]C h o n g S P,J a n g i M S,W a n K L.M o l e c u l a r characterization and expression of a putative ATPase domain from Eimeria tenella[J].J Biochem Mol Biol Biophys,2002,6(2):123-128.
[14]Tan L,Li Y,Yang X,et al.Genetic diversity and drug sensitivity studies on Eimeria tenella field isolates from Hubei Province of China[J].Parasit Vectors,2017,10(1):137.
[15]王泽英,黄金明,王长法,等.荷斯坦牛红细胞Na+-K+-ATP酶的研究进展[J].中国畜牧兽医,2009,36(11):30-37.
[16]阎晓菲,韩红玉,黄兵,等.鸡堆型艾美耳球虫孢子化卵囊cDNA文库的免疫学筛选[J].安徽农业科学,2014,42(14):4363-4364,4454.
[17]阎晓菲.堆形艾美耳球虫孢子化卵囊cDNA文库构建与抗原基因筛选[D].乌鲁木齐:新疆农业大学,2008.
[18]刘铭,孙津红,王凯,等.Bcl-x基因转录后剪切方式改变在大鼠胰岛细胞凋亡中的作用[J].中国糖尿病杂志,2002,10(2):89-92.
[19]崔明亮,李艳,张文成,等.基因组中的基因家族[J].中国老年保健医学,2009,7(2):67-68.
[20]武小霞,王敏,陈庆山,等.大豆再生相关基因GmLEC的克隆及生物信息学分析[J].东北农业大学学报,2015,46(4):1-9.
[21]罗娟,张然,吴柳,等.鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析[J].第三军医大学学报,2018,40(1):23-30.
[22]张经伟,李琦,陈宗艳,等.新型鸭细小病毒NS1基因的原核表达及生物信息学分析[J].中国动物传染病学报,2017,25(2):49-55.
[23]李聪,董辉,朱顺海,等.柔嫩艾美耳球虫胱硫醚β合成酶基因克隆及在真核细胞中的表达[J].中国动物传染病学报,2016,24(2):53-61.
[24]阎晓菲,黄兵,韩红玉,等.鸡堆型艾美耳球虫新基因的克隆与生物信息学分析[J].中国畜牧兽医,2014,41(7):49-53.
[25]D?ppler H,Storz P.A novel tyrosine phosphorylation site in protein kinase D contributes to oxidative stressmediated activation[J].J Biol Chem,2007,282(44):31873-31881.
[26]Lingueglia E,Deval E,Lazdunski M.FMRFamidegated sodium channel and ASIC channels:a new class of ionotropic receptors for FMRFamide and related peptides[J].Peptides,2006,27(5):1138-1152.
[27]陈宁,王平,程田,等.鼠隐藏管状线虫烯醇化酶基因的克隆与序列分析[J].中国动物传染病学报,2017,25(1):50-58.
[28]郑海军,朱荣,葛春蕾,等.人白细胞介素-29的生物信息学分析[J].中国生物制品学杂志,2013,26(2):209-212,217.
[29]朱笠.早发型肌张力障碍相关蛋白质torsinA是一种新型的内质网ATP酶[C]//中国生物化学与分子生物学会.第十届全国酶学学术讨论会论文集.上海:中国生物化学与分子生物学会,2011:43.
[30]钟丰.棉铃虫V-ATP酶A亚基在Cry1Ac杀虫及抗性演化中的作用研究[D].北京:中国农业科学院,2015.
[31]肖晓玲.有氧运动联合补充MT对被动吸烟大鼠抗氧化能力和ATP酶活性的影响研究[D].南昌:江西师范大学,2014.
[32]康自强.盐度胁迫对星洲红鱼生长、消化酶和免疫因子活性及肌肉品质的影响[D].厦门:集美大学,2014.
[33]王泽英,黄金明,王长法,等.荷斯坦牛红细胞Na+-K+-ATP酶的研究进展[J].中国畜牧兽医,2009,36(11):30-37.
[34]Smith C K2nd,Galloway R B.Influence of monensin on cation influx and glycolysis of Eimeria tenella sporozoites in vitro[J].J Parasitol,1983,69(4):666-670.
[35]Wang Z,Suo X,Xia X,et al.Influence of monensin on cation influx and Na+-K+-ATPase activity of Eimeria tenella sporozoites in vitro[J].J Parasitol,2006,92(5):1092-1096.
[36]del Cacho E,Gallego M,Francesch M,et al.Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection[J].Parasitol Int,2010,59(4):506-511.
[37]Bhogal B S,Miller G A,Anderson A C,et al.Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections[J].Vet Immunol Immunopathol,1992,31(3-4):323-335.
[38]Shah M A,Yan R,Xu L,et al.A recombinant DNAvaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E.tenella infection[J].Vet Parasitol,2010,169(1-2):185-189.
[39]Geriletu,Xu L,Xu R H,et al.Vaccination of chickens with DNA vaccine expressing Eimeria tenella MZ5-7 against coccidiosis[J].Vet Parasitol,2011,177(1-2):6-12.
[40]Zhao Y L,Liu Y W,Liu L Y et al.Expression and identification of the ADF-linker-3-1E gene of Eimeria acervulina of chicken[J].Parasitol Res,2016,115(4):1641-1647.
[41]Song X,Zhang R,Xu L,et al.Chimeric DNA vaccines encoding Eimeria acervulina macrophage migration inhibitory factor(E.MIF)induce partial protection against experimental Eimeria infection[J].Acta Parasitol,2015,60(3):500-508.
[42]Song H,Yan R,Xu L,et al.Efficacy of DNA vaccines carrying Eimeria acervulina lactate dehydrogenase antigen gene against coccidiosis[J].Exp Parasitol,2010,126(2):224-231.
[43]Zhu H,Xu L,Yan R,et al.Identification and characterization of a cDNA clone-encoding antigen of Eimeria acervulina[J].Parasitology,2012,139(13):1711-1719.
[44]丛培君.鸡堆型艾美耳球虫乳酸脱氢酶增强型核酸疫苗构建和免疫保护研究[D].哈尔滨:东北农业大学,2014.
[45]黄艺华.鸡堆型艾美耳球虫Rhomboid增强型核酸疫苗构建及免疫保护作用[D].哈尔滨:东北农业大学,2016.
[2]Burt S A,Tersteeg-zijderveld M H,JongeriusGortemaker B G,et al.In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals[J].Vet Parasitol,2013,191(3-4):374-378.
[3]Del Cacho E,Gallego M,Lee S H,et al.Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells(DC)and DC-derived exosomes[J].Vaccine,2011,29(21):3818-3825.
[4]Sun H,Wang L,Wang T,et al.Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis[J].Vaccine,2014,32(16):1869-1876.
[5]Gurunathan S,Wu C Y,Freidag B L,et al.DNAvaccines:a key for inducing long-term cellular immunity[J].Curr Opin Immunol,2000,12(4):442-447.
[6]刘晶,陈复辉,陈淑芳,等.钠钾ATP酶的临床研究进展[J].现代医学,2016,44(6):914-917.
[7]孙董董,林春花,李博勋,等.3种炭疽菌P型ATP酶基因家族分析[J].热带作物学报,2015,36(4):737-745.
[8]盛江筠,朱敏,黄正蔚,等.耐氟菌株及其亲代菌株ATP酶基因的DNA测序[J].上海第二医科大学学报,2005,25(4):340-341.
[9]王竹青,任宪云,高保全,等.三疣梭子蟹F型ATP酶β亚基(F-ATPaseβ)基因的克隆、组织表达及在家系近交中的变化[J].渔业科学进展,2018,39(1):97-106.
[10]马欢.小地老虎V-ATP酶A、B亚基的克隆及B亚基昆虫杆状病毒沉默载体构建的研究[D].杨凌:西北农林科技大学,2015.
[11]陈军营,岳润清,陈新建,等.小麦质膜质子ATP酶基因和启动子序列(登录号:AY829002)[J].分子植物育种,2005,3(6):903-904.
[12]张艳芳,孙瑞芬,郭树春,等.向日葵V-ATPase a3亚基基因的克隆及表达分析[J].中国生物工程杂志,2017,37(5):19-27.
[13]C h o n g S P,J a n g i M S,W a n K L.M o l e c u l a r characterization and expression of a putative ATPase domain from Eimeria tenella[J].J Biochem Mol Biol Biophys,2002,6(2):123-128.
[14]Tan L,Li Y,Yang X,et al.Genetic diversity and drug sensitivity studies on Eimeria tenella field isolates from Hubei Province of China[J].Parasit Vectors,2017,10(1):137.
[15]王泽英,黄金明,王长法,等.荷斯坦牛红细胞Na+-K+-ATP酶的研究进展[J].中国畜牧兽医,2009,36(11):30-37.
[16]阎晓菲,韩红玉,黄兵,等.鸡堆型艾美耳球虫孢子化卵囊cDNA文库的免疫学筛选[J].安徽农业科学,2014,42(14):4363-4364,4454.
[17]阎晓菲.堆形艾美耳球虫孢子化卵囊cDNA文库构建与抗原基因筛选[D].乌鲁木齐:新疆农业大学,2008.
[18]刘铭,孙津红,王凯,等.Bcl-x基因转录后剪切方式改变在大鼠胰岛细胞凋亡中的作用[J].中国糖尿病杂志,2002,10(2):89-92.
[19]崔明亮,李艳,张文成,等.基因组中的基因家族[J].中国老年保健医学,2009,7(2):67-68.
[20]武小霞,王敏,陈庆山,等.大豆再生相关基因GmLEC的克隆及生物信息学分析[J].东北农业大学学报,2015,46(4):1-9.
[21]罗娟,张然,吴柳,等.鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析[J].第三军医大学学报,2018,40(1):23-30.
[22]张经伟,李琦,陈宗艳,等.新型鸭细小病毒NS1基因的原核表达及生物信息学分析[J].中国动物传染病学报,2017,25(2):49-55.
[23]李聪,董辉,朱顺海,等.柔嫩艾美耳球虫胱硫醚β合成酶基因克隆及在真核细胞中的表达[J].中国动物传染病学报,2016,24(2):53-61.
[24]阎晓菲,黄兵,韩红玉,等.鸡堆型艾美耳球虫新基因的克隆与生物信息学分析[J].中国畜牧兽医,2014,41(7):49-53.
[25]D?ppler H,Storz P.A novel tyrosine phosphorylation site in protein kinase D contributes to oxidative stressmediated activation[J].J Biol Chem,2007,282(44):31873-31881.
[26]Lingueglia E,Deval E,Lazdunski M.FMRFamidegated sodium channel and ASIC channels:a new class of ionotropic receptors for FMRFamide and related peptides[J].Peptides,2006,27(5):1138-1152.
[27]陈宁,王平,程田,等.鼠隐藏管状线虫烯醇化酶基因的克隆与序列分析[J].中国动物传染病学报,2017,25(1):50-58.
[28]郑海军,朱荣,葛春蕾,等.人白细胞介素-29的生物信息学分析[J].中国生物制品学杂志,2013,26(2):209-212,217.
[29]朱笠.早发型肌张力障碍相关蛋白质torsinA是一种新型的内质网ATP酶[C]//中国生物化学与分子生物学会.第十届全国酶学学术讨论会论文集.上海:中国生物化学与分子生物学会,2011:43.
[30]钟丰.棉铃虫V-ATP酶A亚基在Cry1Ac杀虫及抗性演化中的作用研究[D].北京:中国农业科学院,2015.
[31]肖晓玲.有氧运动联合补充MT对被动吸烟大鼠抗氧化能力和ATP酶活性的影响研究[D].南昌:江西师范大学,2014.
[32]康自强.盐度胁迫对星洲红鱼生长、消化酶和免疫因子活性及肌肉品质的影响[D].厦门:集美大学,2014.
[33]王泽英,黄金明,王长法,等.荷斯坦牛红细胞Na+-K+-ATP酶的研究进展[J].中国畜牧兽医,2009,36(11):30-37.
[34]Smith C K2nd,Galloway R B.Influence of monensin on cation influx and glycolysis of Eimeria tenella sporozoites in vitro[J].J Parasitol,1983,69(4):666-670.
[35]Wang Z,Suo X,Xia X,et al.Influence of monensin on cation influx and Na+-K+-ATPase activity of Eimeria tenella sporozoites in vitro[J].J Parasitol,2006,92(5):1092-1096.
[36]del Cacho E,Gallego M,Francesch M,et al.Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection[J].Parasitol Int,2010,59(4):506-511.
[37]Bhogal B S,Miller G A,Anderson A C,et al.Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections[J].Vet Immunol Immunopathol,1992,31(3-4):323-335.
[38]Shah M A,Yan R,Xu L,et al.A recombinant DNAvaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E.tenella infection[J].Vet Parasitol,2010,169(1-2):185-189.
[39]Geriletu,Xu L,Xu R H,et al.Vaccination of chickens with DNA vaccine expressing Eimeria tenella MZ5-7 against coccidiosis[J].Vet Parasitol,2011,177(1-2):6-12.
[40]Zhao Y L,Liu Y W,Liu L Y et al.Expression and identification of the ADF-linker-3-1E gene of Eimeria acervulina of chicken[J].Parasitol Res,2016,115(4):1641-1647.
[41]Song X,Zhang R,Xu L,et al.Chimeric DNA vaccines encoding Eimeria acervulina macrophage migration inhibitory factor(E.MIF)induce partial protection against experimental Eimeria infection[J].Acta Parasitol,2015,60(3):500-508.
[42]Song H,Yan R,Xu L,et al.Efficacy of DNA vaccines carrying Eimeria acervulina lactate dehydrogenase antigen gene against coccidiosis[J].Exp Parasitol,2010,126(2):224-231.
[43]Zhu H,Xu L,Yan R,et al.Identification and characterization of a cDNA clone-encoding antigen of Eimeria acervulina[J].Parasitology,2012,139(13):1711-1719.
[44]丛培君.鸡堆型艾美耳球虫乳酸脱氢酶增强型核酸疫苗构建和免疫保护研究[D].哈尔滨:东北农业大学,2014.
[45]黄艺华.鸡堆型艾美耳球虫Rhomboid增强型核酸疫苗构建及免疫保护作用[D].哈尔滨:东北农业大学,2016.