鸡柔嫩艾美耳球虫杂交株F3的培育及微线相关基因研究
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摘要
本实验用本室培育的两个E.tenella 杂交株F2 作为亲本进行杂交,应用RAPD分析方法鉴别出E.tenella 杂交株F3;根据我们实验室已获得的E.tenella 含有部分血栓粘合素及终止子的序列设计引物,利用cDNA 末端快速扩增技术获得一个完整开放阅读框,将其命名为EtM32,应用计算机软件对其序列和功能分析,结果表明该基因与顶复门寄生虫微线蛋白基因有较高同源性,推测其功能与黏附和侵入宿主细胞有关;将EtM32 克隆到pET-28b(+)载体构建成原核表达载体pET-28b-EtM32 并在宿主菌BL21(DE3)得到高效表达,EtM32 重组蛋白经不同免疫途径接种雏鸡,结果表明口服工程活菌组及肌注重组蛋白均可诱导机体的体液免疫反应,以粪便卵囊数评价免疫保护效果,口服工程活菌组的卵囊排出量显著低于对照组(P<0.05),肌注重组蛋白组卵囊排出量与对照组相比差异不显著(P>0.05)。
Coccidiosis, caused by a variety species of the genus Eimeria, is an important disease which harms modern chicken-keeping and exists widely.E.tenella is an important species among those protozoans. Until now a several problems need to be solved like drug-resistance, residual drug, so immunoprophylaxis has become the study focus of coccidiosis. The genus Eimeria includes nine species and there has antigens variation between species, strains and different developmental stages, making available vaccine not completely protect different strains E.tenella. The molecular biology technique has widespread been applicated in Coccidiosis immunoprophylaxis study, but the most important reason which limits the study of E.tenella is that genome project for E.tenella is not completed.
Selection of hybridization parents Single sporocyst from CBG strains and HSC strains, which was separated by micromanipulation technique, were inoculated 1-day-old chicken and five chicken were successfully inoculated. Oocysts were collected respectively and made spores. Their genome DNA was extracted and amplified using random primers. The results demonstrated that CBG7 and CBG11 were two clonal populations which had pathogenicity and immunogenicity. Their protection from themselves rates were 86% and 81% respectively, and their cross protection rates were 65% and 59%. So they were used as hybridization parents in the experiment.
Cultivation of hybrid strain F3 Genetic stability of CBG7 and HSC11 strains were analyzed by comparing genomic fingerprints between the passaged and primary strains. Their immunogenicity were analyzed by OPG counting, stool scoring, cecal lesion scoring, BW assay and protection rate calculating when 10-day-old chicken were inoculated. The 1-day-old chicken was inoculated with the mixed
oocysts including CBG7 and HSC11 strain to get oocyst and single sporocyst was separated to inoculated chicken, hybrid strain F3 , whose pathogenicity and immunogenicity were analyzed, was established. A special fragment was cloned from CBG strain and it only hybridizated with CBG strain and hybrid strain F3, demonstrating the gene has high specificity. Cloning of gene related to microneme from E.tenella A sequence included thrombospondin and terminator was discovered by sequencing of E.tenella. RAPD sequence and it has structure domain characterized by microneme-protein family which was amplified by 5'-RACE and denominated by EtM32. Analysis of Amino acid sequence and protein function demonstrated that the gene was related to micronemes which are important for parasite motility and host cell invasion. Prokaryotic expression of EtM32 related to microneme The recombinant plasmids pET-EtM32 was constructed by cloning EtM32 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host BL21(DE3).The expression levels were optimized by manipulating host strain,the media,the time of harvest. The recombintant proteins were highly expressed in normal LB media, then induced by the 1mmol/L IPTG for 5h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant EtM32 wre up to 22% of the total bacterial protein in the cell lysate. It was specificity of E.tenella by Western-blotting. Immunoprotection of recombinant antigen against E.tenella challenge The chicken were inoculated with recombinant antigen by different pathways, then were challenged with E.tenella oocysts. The results showed recombinant live E.coli strain could induce immunologic reaction and protect partially against E.tenella challenge. Oocysts number and timing shedding of experiment grops were shorter, the increase of body weight (BW) were swifer, and the cecum Lesion were slighter than those of the control . The studies has established foundations for interpreting immunoprotection
Coccidiosis, caused by a variety species of the genus Eimeria, is an important disease which harms modern chicken-keeping and exists widely.E.tenella is an important species among those protozoans. Until now a several problems need to be solved like drug-resistance, residual drug, so immunoprophylaxis has become the study focus of coccidiosis. The genus Eimeria includes nine species and there has antigens variation between species, strains and different developmental stages, making available vaccine not completely protect different strains E.tenella. The molecular biology technique has widespread been applicated in Coccidiosis immunoprophylaxis study, but the most important reason which limits the study of E.tenella is that genome project for E.tenella is not completed.
Selection of hybridization parents Single sporocyst from CBG strains and HSC strains, which was separated by micromanipulation technique, were inoculated 1-day-old chicken and five chicken were successfully inoculated. Oocysts were collected respectively and made spores. Their genome DNA was extracted and amplified using random primers. The results demonstrated that CBG7 and CBG11 were two clonal populations which had pathogenicity and immunogenicity. Their protection from themselves rates were 86% and 81% respectively, and their cross protection rates were 65% and 59%. So they were used as hybridization parents in the experiment.
Cultivation of hybrid strain F3 Genetic stability of CBG7 and HSC11 strains were analyzed by comparing genomic fingerprints between the passaged and primary strains. Their immunogenicity were analyzed by OPG counting, stool scoring, cecal lesion scoring, BW assay and protection rate calculating when 10-day-old chicken were inoculated. The 1-day-old chicken was inoculated with the mixed
oocysts including CBG7 and HSC11 strain to get oocyst and single sporocyst was separated to inoculated chicken, hybrid strain F3 , whose pathogenicity and immunogenicity were analyzed, was established. A special fragment was cloned from CBG strain and it only hybridizated with CBG strain and hybrid strain F3, demonstrating the gene has high specificity. Cloning of gene related to microneme from E.tenella A sequence included thrombospondin and terminator was discovered by sequencing of E.tenella. RAPD sequence and it has structure domain characterized by microneme-protein family which was amplified by 5'-RACE and denominated by EtM32. Analysis of Amino acid sequence and protein function demonstrated that the gene was related to micronemes which are important for parasite motility and host cell invasion. Prokaryotic expression of EtM32 related to microneme The recombinant plasmids pET-EtM32 was constructed by cloning EtM32 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host BL21(DE3).The expression levels were optimized by manipulating host strain,the media,the time of harvest. The recombintant proteins were highly expressed in normal LB media, then induced by the 1mmol/L IPTG for 5h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant EtM32 wre up to 22% of the total bacterial protein in the cell lysate. It was specificity of E.tenella by Western-blotting. Immunoprotection of recombinant antigen against E.tenella challenge The chicken were inoculated with recombinant antigen by different pathways, then were challenged with E.tenella oocysts. The results showed recombinant live E.coli strain could induce immunologic reaction and protect partially against E.tenella challenge. Oocysts number and timing shedding of experiment grops were shorter, the increase of body weight (BW) were swifer, and the cecum Lesion were slighter than those of the control . The studies has established foundations for interpreting immunoprotection
引文
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[2] 谢庆阁, 中和主编, 畜禽重大疫病防治研究进展. 北京; 中国农业科技出版1996 ,181～190.
[3] Rose M.E Immunity to coccidiosis:Interactions in vitro between Eimeria tenella and chicken phagocytic cells. .Biochemistry of Parasites and Host-Parasite Relationships, PP.449-455.
[4] Davis.A mechanism for secretory IgA-mediated inhibition of the cell penetration and intracellular development of Eimeria tenella. Parasitology 1979 36:471-477.
[5] Lillehoj H.S.. JenkinsMC Effects of majorhistocompatibity genes and antien delivery on induction of protectective mucosa limmunity to E.acervulina following immunization with are combinantmerozoite antigen.Immunology.1990,71:1.
[6] Rose,M.E.Identification and characterization of a target antigen of antibody directed against Eimeria tenella merozoites.Immunity to coccidiosis. Parasitology 87 299-313.
[7] H.S.Lillehoj. A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge. Vaccine, 2000,15:243-248.
[8] Talebi.The immunodminant Eimeria acervulina sporozoite antigen previously described as is a 19-kilodal to antigen presenting several Eimeri species. Molecular and biochemical parasitology.1994,63:79-86.
[9] Jeffers,T.K.Genetics of coccidia and the host response.Avian Coccidiosis,1978,57-125.
[10] Jenkins,M.C.,Castle,M.D.,and Danforth,H.D.1991.Protective immunization against the intestinal parasite Eimeria acervulina with recombinant coccidial antigen.Poultry Science,70:539-547.
[11] McDougald.Anti-coccidia drug resistance in the southeastern United States:polyether drugs.Avian Dis,1981,25:606-609.
[12] 谢明权. 柔嫩艾美尔球虫裂殖子免疫原性的研究. 《畜牧兽医学报》1994,25(4),340-345.
[13] Soulbsy. Immune responses in parasitic infections.Florida:Boca Raton,CRC Press, 1989.276-312.
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