Cloning and Expression of the H Protein Gene Fragment of Canine Distemper Virus Nanjing Strain

MO Xiao-jian; GUO Ai-zhen; LU Cheng-ping*

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October 2004

MO Xiao-jian, GUO Ai-zhen and LU Cheng-ping*. Cloning and Expression of the H Protein Gene Fragment of Canine Distemper Virus Nanjing Strain[J]. Virologica Sinica, 2004, 19(5): 487-489.

Citation: MO Xiao-jian, GUO Ai-zhen, LU Cheng-ping*. Cloning and Expression of the H Protein Gene Fragment of Canine Distemper Virus Nanjing Strain .VIROLOGICA SINICA, 2004, 19(5) : 487-489.

犬瘟热病毒南京株H蛋白基因的克隆与表达

1.
南京农业大学农业部动物疫病诊断与免疫重点实验室，江苏南京 210095

摘要

根据发表的犬瘟热病毒(CDV) 参考株Ondetstepoort的序列设计一对引物，以犬瘟热病毒南京株（CDV NJ-15）感染的Vero细胞总RNA为模板，RT-PCR扩增出1.9kb的全长H蛋白基因，双酶切该全长基因，得到1058bp片段，以正确的阅读框架定向克隆于pET-28b(+)中, 然后将重组质粒转化宿主菌RosettaTM，在37℃1.0mmol/L IPTG诱导下获得良好表达。经SDS-PAGE鉴定，表达的融合蛋白质约38kDa，与预期值一致。免疫转印试验显示，该重组蛋白可被CDV Onderstepoort 兔抗血清识别，表明该重组蛋白具备部分抗原性。
- 犬瘟热病毒
- , H蛋白基因
- , 克隆与表达
- , 免疫转印

Cloning and Expression of the H Protein Gene Fragment of Canine Distemper Virus Nanjing Strain

1.
Key Lab of Animal Diease Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing 210095,China

Abstract

One pair of primers was designed and synthesized based on the Haemagglutinin (H) protein gene Sequercein the Onderstepoort strain of Canine distemper virus(CDV). The templates were produced from the reverse transcription reaction, total RNA was isolated from the Vero cells infected with the Nanjing isolate of CDV (CDV NJ-15). The full-length H gene fragment was amplified by polymerase chain reaction (PCR). Digested with EcoR I and Sal I, a 1058bp fragment of the PCR product was cloned into the expression plasmid vector pET-28b (+).The recombinant was transformed into the RosettaTM and induced to express by 1.0m mol/L IPTG at 37℃.The expression product was identified by SDS-PAGE and found to be 38kDa as expected and confirmed by Western blotting with CDV-Onderstepoort rabbit antiserum. The results revealed that the expressed fusion protein in vitro had the critical antigenitic epitopes of H gene of CDV.
- Canine distemper virus
- , H gene
- , Clonging and Expression
- , Western blotting.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expression of the H Protein Gene Fragment of Canine Distemper Virus Nanjing Strain

1. Key Lab of Animal Diease Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing 210095,China

Abstract: One pair of primers was designed and synthesized based on the Haemagglutinin (H) protein gene Sequercein the Onderstepoort strain of Canine distemper virus(CDV). The templates were produced from the reverse transcription reaction, total RNA was isolated from the Vero cells infected with the Nanjing isolate of CDV (CDV NJ-15). The full-length H gene fragment was amplified by polymerase chain reaction (PCR). Digested with EcoR I and Sal I, a 1058bp fragment of the PCR product was cloned into the expression plasmid vector pET-28b (+).The recombinant was transformed into the RosettaTM and induced to express by 1.0m mol/L IPTG at 37℃.The expression product was identified by SDS-PAGE and found to be 38kDa as expected and confirmed by Western blotting with CDV-Onderstepoort rabbit antiserum. The results revealed that the expressed fusion protein in vitro had the critical antigenitic epitopes of H gene of CDV.