柔嫩艾美耳球虫端粒DNA和TERT基因克隆及端粒酶活性测定
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摘要
鸡球虫病(Coccidiosis)是由艾美耳球虫引起以肠道病变为主要特征的细胞内寄生虫病,呈世界性分布,严重危害养鸡业的发展。其中尤以柔嫩艾美耳球虫(Eimeria tenella,E.tenella)引起的危害最为严重。端粒(Telomere)是真核生物染色体末端能够防止染色体末端融合、降解和重组的特殊核蛋白复合体。端粒酶(Telomerase)是一种核糖核蛋白,能以自身携带的RNA为模板,不断合成新的端粒DNA序列添加到染色体末端以维持染色体端粒长度。目前,有关端粒和端粒酶的研究已经成为生命科学研究的热点之一。而寄生虫防治过程中存在的一个非常棘手的问题就是耐药虫体和虫株的不断出现,严重影响抗寄生虫药和驱虫药的效果。以寄生虫的端粒和端粒酶为切入点,进而分离端粒结合蛋白并针对该蛋白设计药物或疫苗无疑为寄生虫病的预防和治疗开辟了新思路。本研究以E.tenella为研究对象,应用单卵囊分离技术成功分离具有较强感染力的地方分离株。在此基础上,成功构建了富含端粒序列文库,进一步通过Southern杂交和酶切筛选阳性克隆,确定了E.tenella端粒DNA重复序列为5’-TTTAGGG-3’。采用CODEHOP方法根据已登录GenBank其他球虫目原虫端粒酶逆转录酶(TERT)基因序列设计简并引物PCR同时结合3’、5’-RACE技术,成功克隆E.tenella TERT 4688bp全长cDNA序列,其中包括4491bp的ORF编码1497个氨基酸,同时含有42bp 5’UTR和152bp 3’UTR以及多聚腺苷酸尾。利用生物信息学分析软件分析表明该基因包含TERT家族的主要特征性基序。最后采用改进TRAP法检测了E.tenella未孢子化卵囊、子孢子和裂殖子端粒酶活性,结果显示子孢子和裂殖子具有较高的端粒酶活性。本研究为进一步深入探索球虫端粒形成、端粒酶活性调控、基因表达和变异、细胞周期调节等机制并针对端粒结合蛋白设计特异抗球虫靶向药物预防和治疗球虫病奠定了基础。
Chicken coccidiosis is an important avian protozoan disease which seriously harm to the poutry industry worldwide. It was caused by various Eimeria spp. parasitized in intestinal mucosal epithelia, which are characterized with enteric pathological lesions. Among various Eimeria spp., E.tenella, which causes caecal coccidiosis, is highly pathogenic. Presently, the control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Telomere is a special nucleoprotein complex located at the ends of chromosomes that protects the termini from fusion, degradation, and recombination. Telomerase is a ribonucleoprotein complex responsible for extending the G-rich strand of telomere repeats composed of RNA and protein subunit. Telomere binding protein can adjust the length by adjusting the activity of telomerase. It has been becoming one of the spotlights of biological research to study on the telomeres and telomerase. The telomere and telomerase of parasites play a vital role in development, gene expression and variation, cell cycle regulation, gene recombination and life cycle composition and so on. E.tenella as the research target in this study, a powerful infectivity strain was isolated from Jilin province by single-oocyst isolation technique and its correlated bionomics was studied. On the basis of it, a libarary including telomeric DNA was constructed successfully. Then the probe was desiged according to orther parasites telomeric DNA and the positive clones which have telomeric DNA were screened from the constructed libarary, thus the E.tenella telomeric DNA was identified. At the same time, pairs of degenerate primer were designed on the basis of other coccidian protozoon TERT sequence and E.tenella TERT full-length cDNA cloned successfully and the composition of E.tenella TERT gene was analyed with protein and nucleic acid sequence analysis software also. At last, the telomerase activity in unsporulated oocyst, sporozoite and merozoite phase was detected using the designed primers according to the infomation of E.tenella telomeric DNA. Data presented in present study provide a biochemical and structural basis for future studies on E.tenella telomerase and will potentially lead to a better understanding of the mechanism of growth and aging in coccidian and in other apicomplexans. The detailed research contents and results as follows:
1. Construction of E.tenella single-oocyst isolation strain
The samples were collected from the serious clinic morbility case which was confirmed as E.tenella infection observation by light microscope. In order to get a single pure line strain to study the E.tenella telometic DNA and TERT sequence, a single-oocyst isolation strain was isolated by single-oocyst isolation technique. Oocyst is ellipsoid, size (21.3±3.2)×(18.9±2.1)μm with a smooth, brown wall, without a micropyle, sporocyst residuum and oocyst residuum,with a polar granule. The sporocysts are oblong ovoid with stieda body. The shortest sporulatic time is 19 h. The minimum prepatent period is 118 h. At the same time, the result of PCR identified that single-oocyst isolation strain was E.tenella. Then the pathogenicity of it was studied, the result of it indicated that the E.tenella has high pathogenic, including their relative weight gain rates can fall 55.2% and the lesion score can reach 3.52, the mortality to chickens can reach 50%, when chickens are inoculated with a dosage of 4.0×104 sporulated oocysts each bird.
2. Identification of telomeric repetitive DNA sequences of E.tenalla
The genomic DNA was extracted from fresh purified E.tenalla oocysts with chloridate and was digested with PstⅠa nd SmaⅠ, the same to pUC18 vector. The digested pruducts were extracted by phenol-chloroform then recoveried ethanol precipitation, the recoveried products was ligated with T4 DNA ligase, the ununited terminal was filled-in with Klenow fragment DNA polymerase I and the products was ligated with 0.1 U T4 DNA ligase at 4℃overnight. The positive clone which has telomeric DNA was screened by Southern blot and restriction enzyme disgestion to sequence. The results of sequencing indicated that 5’-TTTAGGG-3’was the E.tenalla telomeric DNA. In order to indentify the results, oligonucleotide probes (5’-TTTAGGG-3’)5 which was labeled with digoxin were hybridizated with E.tenalla genomic DNA and the E.tenalla genomic DNA which was digested with BAL31 and EcoRⅠin different time. The result of it indicated that the genomic DNA of E.tenalla could hybridizate with oligonucleotide probe and zone of hybridization could be seen clearly. With the time of BAL31 digestion delay, the zone of hybridization on the nylon membrane disappeared. The result showed that the telomeric repetitive DNA sequence of E.tenalla was 5’-TTTAGGG-3’.
3. Cloning and analysis of E.tenella TERT sequence
Degenerate primers were designed based on the sequences of the C.parvum, B.bovis, T.gondii TERT genes. The conserved regions were determined using the BlockMaker program, the degenerate oligonucleotide primers were derived from these conserved sequence blocks with online software CODEHOP. In order to isolate the E.tenella TERT sequence, initially two pairs of degenerate PCR primers were used to amplify a interior fragment of the E.tenella TERT inner sequence. On the basis of the sequence, both 3′and 5′-RACE were performed to isolate upstream and downstream sequences. Sequence analysis of the compiled DNA sequence demonstrated that the full length E.tenella TERT open reading frame encoding a protein of 1497 amino acids with a predicted molecular mass of 172 kDa. Pairwise alignment of full length E.tenella TERT with other previously characterized TERTs using the DNAMAN software revealed high sequence identitities with C.parvum. With conservation of previously identified motifs in the N-terminal region, C-terminal region and the central catalytic domain, motifs GQ, CP and QFP were found within the N-terminal of E.tenella TERT. Like other TERTs, E.tenella TERT contains the previously identified telomerase specific T motif within the central region of the protein followed by RT motifs 1, 2, A, B, C, D and E in the carboxyl terminal half.
4. Detection of telomerase activity E.tenella in different development stages
Fourteen days-old chickens are inoculated with a dosage of 1×105 sporulated oocysts each bird, E.tenella unsporulated oocysts, sporozoites and merozoite were collected. A pairs of primers were desiged according to E.tenella telomeric DNA sequence. The telomerase activity of E.tenella in different development stages were detected by the modified TRAP essay, the telomerase activity of sample which was digested with RNase and CHAPS were detected as negative control. High telomerase activity was detected in sporozoites and merozoites, while not detected in unsporulated oocyst phase.
Chicken coccidiosis is an important avian protozoan disease which seriously harm to the poutry industry worldwide. It was caused by various Eimeria spp. parasitized in intestinal mucosal epithelia, which are characterized with enteric pathological lesions. Among various Eimeria spp., E.tenella, which causes caecal coccidiosis, is highly pathogenic. Presently, the control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Telomere is a special nucleoprotein complex located at the ends of chromosomes that protects the termini from fusion, degradation, and recombination. Telomerase is a ribonucleoprotein complex responsible for extending the G-rich strand of telomere repeats composed of RNA and protein subunit. Telomere binding protein can adjust the length by adjusting the activity of telomerase. It has been becoming one of the spotlights of biological research to study on the telomeres and telomerase. The telomere and telomerase of parasites play a vital role in development, gene expression and variation, cell cycle regulation, gene recombination and life cycle composition and so on. E.tenella as the research target in this study, a powerful infectivity strain was isolated from Jilin province by single-oocyst isolation technique and its correlated bionomics was studied. On the basis of it, a libarary including telomeric DNA was constructed successfully. Then the probe was desiged according to orther parasites telomeric DNA and the positive clones which have telomeric DNA were screened from the constructed libarary, thus the E.tenella telomeric DNA was identified. At the same time, pairs of degenerate primer were designed on the basis of other coccidian protozoon TERT sequence and E.tenella TERT full-length cDNA cloned successfully and the composition of E.tenella TERT gene was analyed with protein and nucleic acid sequence analysis software also. At last, the telomerase activity in unsporulated oocyst, sporozoite and merozoite phase was detected using the designed primers according to the infomation of E.tenella telomeric DNA. Data presented in present study provide a biochemical and structural basis for future studies on E.tenella telomerase and will potentially lead to a better understanding of the mechanism of growth and aging in coccidian and in other apicomplexans. The detailed research contents and results as follows:
1. Construction of E.tenella single-oocyst isolation strain
The samples were collected from the serious clinic morbility case which was confirmed as E.tenella infection observation by light microscope. In order to get a single pure line strain to study the E.tenella telometic DNA and TERT sequence, a single-oocyst isolation strain was isolated by single-oocyst isolation technique. Oocyst is ellipsoid, size (21.3±3.2)×(18.9±2.1)μm with a smooth, brown wall, without a micropyle, sporocyst residuum and oocyst residuum,with a polar granule. The sporocysts are oblong ovoid with stieda body. The shortest sporulatic time is 19 h. The minimum prepatent period is 118 h. At the same time, the result of PCR identified that single-oocyst isolation strain was E.tenella. Then the pathogenicity of it was studied, the result of it indicated that the E.tenella has high pathogenic, including their relative weight gain rates can fall 55.2% and the lesion score can reach 3.52, the mortality to chickens can reach 50%, when chickens are inoculated with a dosage of 4.0×104 sporulated oocysts each bird.
2. Identification of telomeric repetitive DNA sequences of E.tenalla
The genomic DNA was extracted from fresh purified E.tenalla oocysts with chloridate and was digested with PstⅠa nd SmaⅠ, the same to pUC18 vector. The digested pruducts were extracted by phenol-chloroform then recoveried ethanol precipitation, the recoveried products was ligated with T4 DNA ligase, the ununited terminal was filled-in with Klenow fragment DNA polymerase I and the products was ligated with 0.1 U T4 DNA ligase at 4℃overnight. The positive clone which has telomeric DNA was screened by Southern blot and restriction enzyme disgestion to sequence. The results of sequencing indicated that 5’-TTTAGGG-3’was the E.tenalla telomeric DNA. In order to indentify the results, oligonucleotide probes (5’-TTTAGGG-3’)5 which was labeled with digoxin were hybridizated with E.tenalla genomic DNA and the E.tenalla genomic DNA which was digested with BAL31 and EcoRⅠin different time. The result of it indicated that the genomic DNA of E.tenalla could hybridizate with oligonucleotide probe and zone of hybridization could be seen clearly. With the time of BAL31 digestion delay, the zone of hybridization on the nylon membrane disappeared. The result showed that the telomeric repetitive DNA sequence of E.tenalla was 5’-TTTAGGG-3’.
3. Cloning and analysis of E.tenella TERT sequence
Degenerate primers were designed based on the sequences of the C.parvum, B.bovis, T.gondii TERT genes. The conserved regions were determined using the BlockMaker program, the degenerate oligonucleotide primers were derived from these conserved sequence blocks with online software CODEHOP. In order to isolate the E.tenella TERT sequence, initially two pairs of degenerate PCR primers were used to amplify a interior fragment of the E.tenella TERT inner sequence. On the basis of the sequence, both 3′and 5′-RACE were performed to isolate upstream and downstream sequences. Sequence analysis of the compiled DNA sequence demonstrated that the full length E.tenella TERT open reading frame encoding a protein of 1497 amino acids with a predicted molecular mass of 172 kDa. Pairwise alignment of full length E.tenella TERT with other previously characterized TERTs using the DNAMAN software revealed high sequence identitities with C.parvum. With conservation of previously identified motifs in the N-terminal region, C-terminal region and the central catalytic domain, motifs GQ, CP and QFP were found within the N-terminal of E.tenella TERT. Like other TERTs, E.tenella TERT contains the previously identified telomerase specific T motif within the central region of the protein followed by RT motifs 1, 2, A, B, C, D and E in the carboxyl terminal half.
4. Detection of telomerase activity E.tenella in different development stages
Fourteen days-old chickens are inoculated with a dosage of 1×105 sporulated oocysts each bird, E.tenella unsporulated oocysts, sporozoites and merozoite were collected. A pairs of primers were desiged according to E.tenella telomeric DNA sequence. The telomerase activity of E.tenella in different development stages were detected by the modified TRAP essay, the telomerase activity of sample which was digested with RNase and CHAPS were detected as negative control. High telomerase activity was detected in sporozoites and merozoites, while not detected in unsporulated oocyst phase.
引文
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