柔嫩艾美耳球虫2-甲基赤藓糖醇-2，4-环焦磷酸合成酶基因的克隆表达和亚细胞定位研究
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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.
(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
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