| 鸭疫里默氏菌铁依赖抑制子基因的克隆及序列分析 |
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| 引用本文： | 万春和,程龙飞,陈红梅,傅光华,施少华,黄瑜.鸭疫里默氏菌铁依赖抑制子基因的克隆及序列分析[J].中国畜牧兽医,2014,41(12):85-89. |
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| 作者姓名： | 万春和 程龙飞 陈红梅 傅光华 施少华 黄瑜 |
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| 作者单位： | 1. 福建省农业科学院畜牧兽医研究所, 福建省畜禽疫病防治工程技术研究中心, 福建福州 350013;2. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室, 黑龙江哈尔滨 150001 |
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| 基金项目： | 现代农业产业体系建设专项资金(CARS-43);兽医生物技术国家重点实验室开放基金项目(SKLVBF201208). |
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| 摘 要： | 本研究根据鸭疫里默氏菌铁依赖抑制子(RaDtxR)基因序列特征设计特异性引物,利用PCR方法从已鉴定为2型鸭疫里默氏菌RA-FJ株的基因组DNA中扩增目的基因片段,胶回收PCR扩增片段,克隆到pMD18-T载体后进行序列测定.采用生物信息学软件对测序结果进行分析,结果表明所获得的RaDtxR基因编码完整的开放阅读框,全基因长度为654 bp,编码217个氨基酸.所编码的蛋白大小为24.82 ku,理论等电点为5.69,不稳定系数为38.45,属于稳定蛋白质类.将其与GenBank中已发表的RaDtxR基因序列进行核苷酸同源性比对分析,结果显示其与鸭疫里默氏菌疫苗株RA-CH-1株(GenBank登录号:CP003787)的核苷酸同源性为100.0%,与其他4株核苷酸同源性均为99.8%,仅在186位处存在无义突变.利用该基因对福建省农业科学院畜牧兽医研究所分离鉴定的鸭大肠杆菌、禽多杀性巴氏杆菌和沙门氏菌进行扩增,结果显示均未扩增到条带,对福建省农业科学院畜牧兽医研究所分离鉴定的1型、2型、3型、11型和13型鸭疫里默氏菌均可扩增出特异性目的条带,表明RaDtxR基因可作为鸭疫里默氏菌鉴别诊断的靶基因.试验结果表明,本研究成功克隆到鸭疫里默氏菌RaDtxR基因,为研究其功能奠定基础.
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| 关 键 词： | 鸭疫里默氏菌 铁依赖抑制子基因 克隆 生物信息学分析 |
| 收稿时间： | 2014-06-12 |
Cloning and Sequence Analysis of RaDtxR Gene from Riemerella anatipestifer |
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| WAN Chun-he,CHENG Long-fei,CHEN Hong-mei,FU Guang-hua,SHI Shao-hua,HUANG Yu.Cloning and Sequence Analysis of RaDtxR Gene from Riemerella anatipestifer[J].China Animal Husbandry & Veterinary Medicine,2014,41(12):85-89. |
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| Authors: | WAN Chun-he CHENG Long-fei CHEN Hong-mei FU Guang-hua SHI Shao-hua HUANG Yu |
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| Institution: | 1. Fujian Animal Disease Control Technology Development Center, Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Science, Fuzhou 350013, China;2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China |
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| Abstract: | A polymerase chain reaction method was used to amplify the RaDtxR gene from the identified Riemerella anatipestifer serotype 2 RA-FJ strain, the target fragment was then cloned and sequenced, which included the encoding open reading frame (ORF) of RaDtxR gene. The nucleotide sequence and deduced amino acid sequence were analyzed by the bioinformatics software. The cloned sequence was 654 nucleotides in length, coding an ORF with 217 amino acids. The molecular weight, theoretical isoelectric point and instability index of the Riemerella anatipestifer RaDtxR gene were 24.82 ku, 5.69 and 38.45, respectively. The cloned RaDtxR gene shared the highest homology with Riemerella anatipestifer vaccine strain RA-CH-1 (GenBank accession number was CP003787), with 100.0%. Also, it shared 99.8% with other 4 Riemerella anatipestifer RaDtxR genes, which shared only a nonsense mutation at the position 186. No amplification was detected from other bacteria or virus, such as duck E.coli, avain Pasteurella multocida and Salmonella, the PCR results of Riemerella anatipestifer serotypes 1, 2, 3, 11, 13 were positive, which demonstrated that RaDtxR gene could be used as a diagnosis target gene for Riemerella anatipestifer.The results showed that we had successfully cloned the RaDtxR gene from Riemerella anatipestifer, which would lay a foundation for research of its function. |
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| Keywords: | Riemerella anatipestifer RaDtxR gene clone bioinformatics analysis |
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