Abstract
The high genetic variability of RNA viruses is a significant factor limiting the discovery of effective biomarkers, the development of vaccines, and characterizations of the immune response during infection. Protein microarrays have been shown to be a powerful method in biomarker discovery and the identification of novel protein–protein interaction networks, suggesting that this technique could also be very useful in studies of infectious RNA viruses. However, to date, the amount of genetic material required to produce protein arrays, as well as the time- and labor-intensive procedures typically needed, have limited their more widespread application. Here, we introduce a method, protein microarray fabrication through gene synthesis (PAGES), for the rapid and efficient construction of protein microarrays particularly for RNA viruses. Using dengue virus as an example, we first identify consensus sequences from 3,604 different strains and then fabricate complete proteomic microarrays that are unique for each consensus sequence. To demonstrate their applicability, we show that these microarrays can differentiate sera from patients infected by dengue virus, related pathogens, or from uninfected patients. We anticipate that the microarray and expression library constructed in this study will find immediate use in further studies of dengue virus and that, more generally, PAGES will become a widely applied method in the clinical characterization of RNA viruses.
Highlights
From the ‡Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine, Ministry of Education, ¶School of Biomedical Engineering, **State Key Laboratory of Oncogenes and Related Genes,Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China; §Institute of Pathogenic Microbiology, Center for Disease Control and Prevention of Guangdong, No 160, Quanxian Road, Dashi Street, Panyu District, Guangzhou, 511430, China; National Research Institute for Health and Family Planning, No.12 Dahuisi Road, Haidian District, Beijing, 100081, China
We have developed protein microarray fabrication through gene synthesis (PAGES), a new strategy that combines consensus sequence identification, gene synthesis and high-throughput protein purification for the rapid and efficient construction of protein microarrays
We have demonstrated the effectiveness of this method using dengue virus as an example, fabricating the first proteomic microarray covering all four serotypes of this virus in one month
Summary
Many viruses have been a consistent global threat for many years, such as dengue virus that infects tens of millions of people worldwide each year, of which 500,000 develop hemorrhagic fever and 20,000 die [4]. To study these viruses, it is typically necessary to obtain their genetic material. All of the genes/predicted ORFs are PCR amplified with primers containing proper restriction endonuclease sites/recombination sites [7], which can prove challenging for genes with high GC content [8, 9], and cloned into expression vectors This entire procedure is extremely labor- and time-consuming, generally requiring 3– 4. The abbreviations used are: SARS, severe acute respiratory syndrome; PCR, polymerase chain reaction; ORF, open reading frame; ELISA, enzyme linked immunosorbent assay; GST, glutathione Stransferase; PMD, protein microarray database
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