High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein

GE Fei-fei; QIU Ya-feng; YANG Yao-wu; CHEN Pu-yan

PDF
Cite
Share

facebook

twitter

google

linkedin

April 2005

GE Fei-fei, QIU Ya-feng, YANG Yao-wu and CHEN Pu-yan. High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein[J]. Virologica Sinica, 2005, 20(2): 117-120.

Citation: GE Fei-fei, QIU Ya-feng, YANG Yao-wu, CHEN Pu-yan. High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein .VIROLOGICA SINICA, 2005, 20(2) : 117-120.

乙型脑炎病毒SA14-14-2株E蛋白主要抗原片段的高效表达

葛菲菲 ¹ ,
邱亚峰 ¹ ,
杨耀武 ² ,
陈溥言 ^1*

1.
1.南京农业大学农业部动物疫病诊断与免疫重点开放实验室江苏南京 2.中国预防医学科学院病毒研究所北京

摘要

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14 14 2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZαA,以电穿孔法转化酵母X 33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS PAGE和免疫印迹分析表达产物。由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性。在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据。
- 乙脑疫苗株SA14142株
- , E蛋白基因主要抗原片段
- , 巴斯德毕赤酵母
- , 高效表达

High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein

1.
1. Key L aboratory of A nimal Diseases Diagnosis & Immunology of China’s Department of A griculture , Nanj ing A gricultural Univer2 sity , Nanj ing 210095 , China

Abstract

Based on a constructed prokaryotic expression plasmid which carries a ma-jor antigenic segment of E gene of JEV strain SA14-14-2 in our lab, we expressed the gene in methylotrophic yeast Pichia pastoris. The segment was choosn based on an- alysis provided by a computer software because of its higher antigenic index. The seg- ment was 1113bp, encoding 371 aa. The gene was subcloned into the vector pPICZα-A. The constructed plasmid was transformed into yeast X-33 by electrophoration. The recombinant transformants were selected by Zeocin. Induced by the addition of metha- nol every 24h, the product analyzed by SDS-PAGE was about 44kDa and 50kDa at a yield of 290 mg per litter of culture. The result of Western blotting indica- ted that the two bands both have specific antigenicity mainly owing to heterogeneous glycosylation. Coating with the supernatant, we can easily discriminate the positive serum from the negative, which should be useful for the production of diagnostic agents and genetically engineered vaccine vaccine of J EV.
- Japanese Encephalitis virus
- , SA 14-14-2 strain
- , Major antigenic segment of E protein
- , Pichia pastoris

References
Proportional views

PDF

Article Metrics

Article views(4202) PDF downloads(1017) Cited by(0)

Proportional views

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

High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein

1. 1. Key L aboratory of A nimal Diseases Diagnosis & Immunology of China’s Department of A griculture , Nanj ing A gricultural Univer2 sity , Nanj ing 210095 , China

Abstract: Based on a constructed prokaryotic expression plasmid which carries a ma-jor antigenic segment of E gene of JEV strain SA14-14-2 in our lab, we expressed the gene in methylotrophic yeast Pichia pastoris. The segment was choosn based on an- alysis provided by a computer software because of its higher antigenic index. The seg- ment was 1113bp, encoding 371 aa. The gene was subcloned into the vector pPICZα-A. The constructed plasmid was transformed into yeast X-33 by electrophoration. The recombinant transformants were selected by Zeocin. Induced by the addition of metha- nol every 24h, the product analyzed by SDS-PAGE was about 44kDa and 50kDa at a yield of 290 mg per litter of culture. The result of Western blotting indica- ted that the two bands both have specific antigenicity mainly owing to heterogeneous glycosylation. Coating with the supernatant, we can easily discriminate the positive serum from the negative, which should be useful for the production of diagnostic agents and genetically engineered vaccine vaccine of J EV.