Development And Application Of Diagnostic ELISA-Array Protein Chip For Detection Antibodies Against Six Arboviruses And Streptococcus Suis Serotype 2

In 21th century, infectious diseases are still the major causes of human death. With the ancient infectious diseases existing, the emerging, re-emerging infectious diseases and the drug-resistant pathogens become big challenges to human health. Moreover, as the threat of bio-terrorism increases, bio-defense has been a big problem for the world. A pristine detection and diagnosis of diseases is crucial for handling emerging infectious diseases and bio-terrorism. The widespread zoonosis pathogens such as Dengue virus, Tick-borne encephalitis virus, Japanese encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Yellow fever virus, West Nile virus and Streptococcus suis serotype 2 have been great hazards for society health. Thus, the construction of a rapid and high-throughput platform to detecting the infectious pathogens is of great importance.This subject aims at the construction of a high-throughput and new platform based on ELISA-array to dectect the important pathogens. As an efficient supplement to the existing methods, this subject will ensure the rapid detection of pathogens when the disease emerges, and can enhance the new pathogen detection capability of our country. The primer pairs for amplifying the genes were designed based on the whole genomic sequence of bacteria and viruses (DENV、TBEV、JBEV、EEEV、WEEV、YFV、WNV、SS2, etc) provided by GenBank. High conserved region of the genes were selected for primer designing according to the prediction results of the bioinformatics software (BioEdit, Invitrogen, etc) and the restriction enzymes sequences were added to the primers which allow the PCR products to be recombined with the expression plasmid. The amplified gene targets were cloned into pET-30a and pET-32a, with recombinant proteins successfully expressed as His (6)-tagged fusion proteins in E. coli BL21.1. SDS-PAGE analysis of the expression product and micoarrays preparationThe recombinant proteins were purified by affinity chromatography with Ni-NTA resin. Then they were printed onto the 96 wells of polystyrenes material plates by the robot of Bio-Dot AD5000TM. The printed plates were blocked in PBS supplemented with 10%BSA for 1 h, then deposited at room temperature overnight and stored at 4℃. 2. Screening out recombinant proteins by probing microarryNine of 16 viral proteins and 5 of 23 Streptococcus suis serotype 2 proteins that meeting the specificity and sensitivity standard were screened out by probing microarry with immunized rabbit polyclonal antisera and detecting the specific antibody level. The identified antigens were tested by enzyme-linked immunosorbent assay (ELISA) and the sensitivity and specificity of the ELISA-array platform were also evaluated. As a result, the dectection range of ELIS A-array is 1:6400, which was 4-16 times more sensitive than the traditional ELISA tests. For further study, the 23 Streptococcus suis serotype 2 antigens were probed with sera from immunized or infected pig. Antigens ssu253、ssu934、ssu1476、ssu186、ssu1355�'�MRP were found to have significant difference between the sera before and after immunity(P<0.01), and ssu1000、ssu934、ssu1476、ssu186、ssul664、ssu1355、MRP were found to have significant difference between the sera before and after infection (P<0.01).3. Clinical serum samples detectionIn summary, our study has developed a multiplex ELISA based protein array for detection of antibodies against the 6 arboviruses viruses and Streptococcus suis Serotype 2. We also optimizaed the aspects of the platform (including store life、reaction conditions、dectection range、samples treatment, etc) and normalized the procedure, which will pave the way for the construction of a better micrroarray platform. Horseradish peroxidase-conjugated goat anti-human immunoglobulin G and immunoglobulin M were used to detect the antibodys of clinical serum samples in order to distinguish the phases of immunity response, viral antigens DEN-E-D3、DEN2-E-D3、TBE-E-D3 and Streptococcus suis serotype 2 antigens ssu934、ssu186、ssu1664、ssu1355、MRP were proved to have significant difference between the sera before and after human infection.