| 摘要: |
| 【目的】对柔嫩艾美耳球虫（E.tenella）杨凌株RB1-a基因进行克隆与表达，检测表达产物的免疫保护性，为鸡球虫基因工程疫苗的研制奠定基础。【方法】以纯化的E.tenella杨凌株孢子化卵囊的总RNA为模板，用RT-PCR方法扩增出其RB1-a基因，测序后进行序列分析。然后将RB1-a基因N端不含信号肽序列克隆到表达载体pET-32a(+)中，构建pET-32a-RB1-a重组质粒，并在大肠埃希菌BL21(DE3)中诱导表达出重组蛋白，将重组蛋白纯化后免疫雏鸡，对其免疫效果进行评价。【结果】柔嫩艾美耳球虫杨凌株RB1-a基因的开放阅读框（ORF）包含618个碱基，编码205个氨基酸，与GenBank报道的柔嫩艾美耳球虫PAPa46株ORF序列相似性为99.84%，两者推导的氨基酸序列相同。SDS-PAGE分析表明，重组质粒表达分子质量为42 ku的融合蛋白。免疫效果测定结果显示，RB1-a免疫组和RB1-a+佐剂免疫组的相对增重率分别为64.2%和69.4%，卵囊减少率分别达37.22%和30.56%，抗球虫指数分别为146.2和150.4。【结论】RB1-a基因具有很强的保守性，种间差异不大；RB1-a重组蛋白具有一定的免疫保护效果。 |
| 关键词: 柔嫩艾美耳球虫杨凌株 RB1-a基因 克隆 表达 免疫保护 |
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| 基金项目:家畜疫病病原生物学国家重点实验室开放基金项目（SKLVEB2009KFKT7003） |
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| Studies on cloning and expression of RB1-a gene of Eimeria tenella YL strain and immune protection of expressed products in chickens |
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| Abstract: |
| 【Objective】The research studied the cloning and expression of RB1-a gene of E.tenella YL strain and its immune protetion in chickens.【Method】E.tenella RB1-a gene was cloned from the sporozoites of porulated oocysts of by RT-PCR.The recombinant expression plasmid pET-32a-RB1-a was constructed and expressed in BL21(DE3) strain of E.coli.The expressed products were analyzed by SDS-PAGE.The recombinant proteins were used in chickens infected with E.tenella YL strain to judge the protective effect.【Result】The sequence analysis of RB1-a gene of E.tenella LY strain suggested that the ORF of RB1-a gene consisted of 618 nucleotides and encoded 205 amino acids.Compared with the E.acervulina PAPa46 strain,the sequence homologies of nucleotides and deduced amino acids of RB1-a gene was 99.84% and 100%,respectively.SDS-PAGE showed that the fusion protein was successfully expressed and the molecular weight of the protein was 42 ku.The immune protective experiments showed that RB1-a immunization and RB1-a and adjuvant immunization’s relative weight gain rate was 64.2% and 69.4%,oocysts output deduction rate 37.22% and 30.56%,ACI 146.2 and 150.4.【Conclusion】RB1-a gene was highly reserved,and little difference existed between the various species.RB1-a protein had certain protective effect against coccidiosis. |
| Key words: Eimeria tenella YL strain RB1-a gene cloning expression immune protection |
