猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立

doi:10.16431/j.cnki.1671-7236.2017.08.007

Abstract

Abstract:

This study was aimed to construct stable cell line expressing N protein coded by porcine epidemic diarrhea virus (PEDV) strain CH-HuN1301. N gene of this virus strain was amplified by RT-PCR, which was then cloned into prokaryotic expression vector pET28a and eukaryotic expression vector pEGFP-C1,respectively. Recombinant plasmids were sequenced and analyzed by software Mega 5.0. To prepare polyclonal antibody of N protein, the prokaryotic expression recombinant N protein was used as antigen to immunize rabbits. HEK293 transfected with recombinant eukaryotic expression plasmids pEGFP-PEDV-N was selected by G418 to produce a stable cell line which then was identified by immunoperoxidase monolayer assay (IPMA) and fluorescence observation. Results showed that, the N gene of PEDV strain CH-HuN1301 was 1 323 bp, molecular weight of recombinant prokaryotic expressing N protein was about 60 ku, prepared polyclonal antibody against N protein showed wonderful reactivity with PEDV propagated in Vero cells, recombinant eukaryotic expressing N protein was positively detected in the established stable cell line by IPMA and fluorescence observation. This stable cell line provided foundation for the research in PEDV diagnosis methods.

Key words: porcine epidemic diarrhea virus (PEDV); CH-HuN1301; N gene; stable cell line

CLC Number:

S852.65

TANG Qing-hai, YANG Hai, LI Lu, CHEN Guo-liang, HE Li-fang, LIU Zui, WANG Fang-yu. Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301[J]. , 2017, 44(8): 2261-2268.

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Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301

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