| The Rubellavirus (RV) is atogavirus and the sole representative of the Rubivirus subgroup. The Rubella virus causes a relatively mild childhood disease (German measles) which usually results in permanent immunity thought to be mediated by both T-lymphocytes and antibodies. Possible consequences of Rubella infections in adults are transientlchronic arthritis, musculoskeletal syndromes, insulin-dependent diabetes mellitus and late onset of neurologic sequelae. The rubella virus is a main parameter during early pregnancy. Maternal Rubella virus infection during pregnancy is associated with a risk of congenital rubella syndrome (CRS) in the fetus. For this reason, the prevention of congenital abnormalities caused by RV infection during early pregnancy requires the determination of an individual's immune status by serology, that means a selective determination of IgG and IgM antibodies specific for Rubellavirus antigens. The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism. In particular E1 plays a pivotal role in the fusion of the virus with the endosomal membrane. Both glycoproteins are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems.Objects: To construct, express and purify a high-yield overproduction of an engineered E1 ectodomain in the E.coli cytosol, which can be renatured simply and conveniently into a highly soluble and immunoreactive conformation.Methods: We got the artificial coding sequence of ssRVEl variant that encodes two-unit SlyD (aa 1～165) and RV E1 ectodomain (aa 201～432). It was inserted into pET-24a (+), a kind of prokaryotic expression vector, and induced to express by IPTG in E. coli., BL-21(DE3) strain. The protein of ssRVEl variant was purified by immobilized metal ion affinity chromatography (IMAC) and renatured on the column. At last, we analyzed the immunologic characters of the recombinant antigen by ELISA.Results: The renatured fusion protein ssRVEl variant significantly improved solubility, stability and detection sensibility of anti-RV IgG in ELISA assay. However, false positive rate was increased. To enhance expression, facilitate in vitro refolding, and improve the overall solubility of Rubella E1 ectodomain (aa 201～432) variant, two units of the E. coli chaperone SlyD (aa 1～165) was fused to the N-terminal of E1 protein. In fact, our results showed that the sera of several individuals can react with SlyD , and that may be the reseaon of enhanced false positive rate in ELISA assay. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity.Conclusions: The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance. As part of this fusion protein, the E1 ectodomain fragment of residues 201～432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. |
