Cloning，Sequencing and Expression of S Gene Encoding Region 0f Han taan Virus Strain Chen

Xue-Xiao-Beng; XU Zhi-Kai; MA Wen-Yu; YAN Yan; YIN Wen; ZHANG Fang-Lin; TUN Xin-An; DIAO Qian; BAI Wen-Chao

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February 1999

Xue-Xiao-Beng, XU Zhi-Kai, MA Wen-Yu, YAN Yan, YIN Wen, ZHANG Fang-Lin, TUN Xin-An, DIAO Qian and BAI Wen-Chao. Cloning，Sequencing and Expression of S Gene Encoding Region 0f Han taan Virus Strain Chen[J]. Virologica Sinica, 1999, 14(1): 48-51.

Citation: Xue-Xiao-Beng, XU Zhi-Kai, MA Wen-Yu, YAN Yan, YIN Wen, ZHANG Fang-Lin, TUN Xin-An, DIAO Qian, BAI Wen-Chao. Cloning，Sequencing and Expression of S Gene Encoding Region 0f Han taan Virus Strain Chen .VIROLOGICA SINICA, 1999, 14(1) : 48-51.

汉坦病毒陈株S基因编码区的克隆、序列分析及表达

1.
第四军医大学微生物学教研室．西安710032

摘要

从汉坦病毒陈株感染的ＶｅｒｏＥ６细胞裂解液中提取病毒ＲＮＡ，经逆转录ＰＣＲ获得病毒Ｓ基因编码区约１．３ｋｂｃＤＮＡ片段，克隆该片段后进行核苷酸序列测定，并与汉坦病毒７６１１８株进行同源性比较，结果二者核苷酸序列同源性为８６％，推导的氨基酸序列同源性为９７％。将该基因片段插入原核表达载体ｐＧＥＸ４Ｔ１，在大肠杆菌中获得高效表达。表达产物为ＧＳＴＮＰ融合蛋白。ＳＤＳＰＡＧＥ检测表达蛋白分子约７２ｋＤ左右。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ和ＥＬＩＳＡ试验结果表明，表达产物可与多株抗汉坦病毒核蛋白的ＭｃＡｂ发生反应，其抗原表位及ＭｃＡｂ反应谱与７６１１８株相比存在某些差异。
- 汉坦病毒
- , S基因
- , 核苷酸序列分析
- , 基因表达

Cloning，Sequencing and Expression of S Gene Encoding Region 0f Han taan Virus Strain Chen

1.
Dept Microhldogy,The Fourth Military Medical Uniersity,Xi'an 710032

Abstract

RNA of Hantaan virus (HNTV) strain Chen isolated from China was extracted from lysate of Vero E 6 cell infected by the virus. With the RNA as template, 1.3kb cDNA fragment containing the region encoding nucleocapsid protein (NP) was obtained by reverse transcription polymerase chain reaction (RT PCR). This fragment was cloned into pGEM 7zf(+) plasmid and sequenced. Homology comparision showed that the homology of the nucleotide and amino acid sequences between strain Chen and strain 76 118 was 86% and 97%, respectively. The RT PCR product was cloned into pGEX 4T 1 vector and was efficiently expressed in E.coli . The expressed product is NP GST fusion protein with molecular weight about 70 kD in SDS pAGE. Its antigenic epitope was recognized by Western blotting and ELISA using the McAbs. The results showed that there were some differences on antigenic epitopes between strain Chen and strain 76 118.
- Hantaan virus
- , S gene Nucleotide
- , sequencing
- , Expression

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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，Sequencing and Expression of S Gene Encoding Region 0f Han taan Virus Strain Chen

1. Dept Microhldogy,The Fourth Military Medical Uniersity,Xi'an 710032

Abstract: RNA of Hantaan virus (HNTV) strain Chen isolated from China was extracted from lysate of Vero E 6 cell infected by the virus. With the RNA as template, 1.3kb cDNA fragment containing the region encoding nucleocapsid protein (NP) was obtained by reverse transcription polymerase chain reaction (RT PCR). This fragment was cloned into pGEM 7zf(+) plasmid and sequenced. Homology comparision showed that the homology of the nucleotide and amino acid sequences between strain Chen and strain 76 118 was 86% and 97%, respectively. The RT PCR product was cloned into pGEX 4T 1 vector and was efficiently expressed in E.coli . The expressed product is NP GST fusion protein with molecular weight about 70 kD in SDS pAGE. Its antigenic epitope was recognized by Western blotting and ELISA using the McAbs. The results showed that there were some differences on antigenic epitopes between strain Chen and strain 76 118.