Rapid Production of Virus Protein Microarray Using Protein Microarray Fabrication through Gene Synthesis (PAGES)

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.

Fig. 1.

Schematic diagram of PAGES. The coding sequences of the virus of interest were retrieved from NCBI or other database. After the consensus sequences were generated, they were subjected for sequence optimization and gene synthesis. The proteins were then expressed in E. coli and affinity purified. The protein microarrays were fabricated and adopted for biology study and clinical research.

Fig. 2.

The workflow of constructing expression library for dengue virus.

Fig. 3.

Dengue virus proteins of all the four serotypes were successfully purified using a high-throughput way. (A) The synthesized genes were integrated into the expression vector pGEX-4T-1. (B) Expected molecular weight (MW) of elected proteins. (C) The proteins were expressed and purified in a high-throughput format. The E. coli expression clones were stored in a 96-well plate. The clones were inoculated to a 12-channel plate for culture and protein expression. The cells were harvested, and the proteins were affinity purified in a 96-deep well plate. The purified proteins were stored at −80 °C prior to microarray printing. (D) All the purified proteins were validated by Western blotting. The red asterisk indicated the target proteins.

Fig. 4.

Characteristics of the dengue virus proteome microarray. (A) The layout of the proteome microarray. (B) The microarray was incubated with an anti-GST antibody followed with secondary antibody G-A-R-Cy5. (C) Histogram analysis of fluorescence intensity of all the purified proteins according to the signal of secondary antibody G-A-R-Cy5. (D) The microarray was incubated with a secondary antibody M-A-H-IgG-549. (E) The microarray was incubated with a secondary antibody d-A-H-IgM-649. (F) The correlation between the concentration of the serially diluted IgG (left) and IgM (right) on the microarray and corresponding fluorescence intensities.

Fig. 5.

Probing serum on the dengue virus proteome microarray. The microarray signal intensities were transformed to the serum antibody concentration according to the standard curve in each block. Heatmaps were generated based on the data for [IgG] (A) and [IgM] (B) OP1, OP2, OP3 indicated the sera that were collected from patients diagnosed as viral hemorrhagic fever or infected by Scrub typus and pidemic encephalitis B, respectively. (C) Candidate proteins with p value < 0.05 and fold change > 1.

Fig. 6.

Validation of proteins I-E and III-E as potential markers by ELISA. (A) Purified protein I-E (left) and III-E (right) were assessed by silver staining. (B) The phylogenetic relationship of the four protein E. (C) ELISA was performed by using an independent set of samples against protein I-E (left) and III-E (right).

Fig. 7.

The dynamics of [IgG] specific against I-E and III-E. (A) Information of the three patients with sera drawn at different time points from day 2 to day 16. (B–D) The dynamics of [IgG] in serum that bound I-E and III-E for patient 1# (B), patient 2# (C), and patient 3# (D).

Fig. 8.

The comparison the PAGES and the traditional strategy for protein microarray construction.