Abstract
The 24S subgenomic mRNA of rubella virus (RV) specifies a polyprotein which is post-translationally processed to three structural proteins capsid , E2 and E1. E1 and E2 are membrane glycoproteins forming the virion spikes. In the polyprotein, E1 and E2 are both preceded by stretches of uncharged hydrophobic amino acids which were identified as putative signal peptides mediating the translocation of the proteins into the endoplasmic reticulum. The translocation of the glycoprotein is halted by other hydrophobic regions at the carboxy-terminus of both proteins which functions as an anchor. cDNA clones encoding E1 glycoprotein coding region were produced by reverse transcriptase of rubella enhanced mRNA from virus containing supernatant of BHK cells infected with Thomas strain of RV. The nucleotide sequences of both E2 and E1 cDNAs were determined and compared to published sequences from other RV strains. Sequences coding for all or part of the E1 and E2 proteins were inserted into plasmids PUEX1, 2 and 3 for expression in E. coli to produce fusion proteins with where the CDNA fused to the 3' end of the lacZ gene to produce the whole of the E1 protein with subfragments were produced in E. coli in large amounts, (more than 20% of the total cell protein), and proved to be antigenic when reacted with human antisera. The fusion proteins were produced in inclusion bodies. The recombinant antigen β-gal-E1 was used in ELISA and MACRIA diagnosis techniques to detect RV-infected sera, significant results were obtained when IgM anti-rubella antibody was detected. Expression of cDNA fragments coding for all of E2 and subfragments in E. coli had a severe effect on the inhibition of cell growth. Results suggest that the lethal effect could be due to the presence of hydrophobic domains in the protein.The antigens produced in E. coli are unglycosylated and may therefore not be the most appropriate substrates for recombinant vaccine development. Therefore, another approach was pursued for the production of glycosylated products of rubella antigens E1 and E2 by expression in mammalian COS cells. Attempts were made to produce secreted proteins from both E1 and E2. As the putative signal peptide of E1 is not defined, a truncated hydrophobic amino terminal domain (signal peptide) was used to study if the remaining portion could translocate a truncated E1 glycoprotein through the endoplasmic reticulum (ER). E1 was secreted into the media, but not efficiently. The E2 cDNA clone was expressed intracellularly and the removal of half its anchor region at the carboxy-terminus did not result in a secreted protein and had no effect on the protein translation efficiency.