Preperation of antibodies against soluble recombinant dengue E proteins fused with glutathione's transferase / Jiali Wang, Na Gao, Wei Chen, Dongying Li, Limei Liu, Yingjie Wan, Jing An

The envelope glycoprotein (E) of dengue (DENV) viruses mediates viral attachment and entry through membrane fusion. In this study, three fragments (domain 3a, domain 12 and domain 123a) of DENV envelope protein were inserted into plasmid pGEX-6p-1 to express glutathione's transferase (CST) fusion proteins in Escherichia coli (E. coli) strain BL21. High yields of soluble recombinant proteins were obtained as follows: 20 mg/l of GST-Ela protein, 5 mg/L of GST-E12 protein, and 3 mg/t of GST-E123a protein. Although they all failed to protect vero cells from DENV infection in virus-binding blocking assay, three proteins could be recognized by mouse serum against DENV in westem blot analysis. Then, polyclonal antibodies against either GST-E12 or GST-E3a were raised in New Zealand rabbits, and used in immunofluorescence staining. The infected vero cells showed typical cytoplasmatic fluorescence near the nucleus where the virus replication took place. The study indicated that soluble GST fusion E proteins might lack some biological activities of natural dengue E protein. But polyclonal antibodies against GST-E3a and GST-E12 might be helpful tools for further study of DENV pathogenesis in vitro.

Nursing.