Development of a yeast two-hybrid screen for selection of A/H1N1 influenza NS1 non-structural protein and human CPSF30 protein interaction inhibitors.
- Author:
Jian-qiang KONG
1
;
Jun-hao SHEN
;
Yong HUANG
;
Ren-yu RUAN
;
Bin XIANG
;
Xiao-dong ZHENG
;
Ke-di CHENG
;
Wei WANG
Author Information
1. Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC & Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Health of PRC, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Binding Sites;
Cleavage And Polyadenylation Specificity Factor;
genetics;
metabolism;
Drugs, Chinese Herbal;
pharmacology;
Gene Amplification;
HeLa Cells;
Humans;
Influenza A Virus, H1N1 Subtype;
genetics;
Peptide Fragments;
genetics;
Plasmids;
Protein Binding;
drug effects;
Transformation, Genetic;
Two-Hybrid System Techniques;
Viral Nonstructural Proteins;
genetics;
metabolism
- From:
Acta Pharmaceutica Sinica
2010;45(3):388-394
- CountryChina
- Language:Chinese
-
Abstract:
Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3'-end processing of cellular pre-mRNAs by binding the cellular protein: the 30-kDa subunit of CPSF (cleavage and polyadenylation specificity factor, CPSF30). CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction (PCR), and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including 'Shuanghuanglian oral liquid', showed the strong inhibition of the NS1-CPSF30 interaction.
