| Rubella virus (RV), also known as German measles virus, is the only member of Section Togavirus rubella virus, and non-cross-virus antigen with others. RV is one of the TORCH syndrome pathogens (cytomegalovirus, herpes virus, rubella virus, Toxoplasma gondii), one important factor resulted in fetal teratogenicity. The fetus can be infected through the placenta, resulting in newborns suffering from congenital rubella syndrome(CRS), appeared to be cataracts, deafness, heart disease or mental retardation. The sooner RV infection is in pregnant women, the greater newborns suffering from CRS. RV is also the impact factor of male infertility. TORCH pathogens infected in male reproductive tract can cause orchitis, epididymitis, prostatitis, as well as male infertility.Currently, immunological methods have been widely used in clinical diagnosis of RV infection, such as hemagglutination inhibition test (HIA), the assay of specific antibodies IgM, IgG to RV and the detection of RV antigen and other proteins. The screening of RV infection is mainly based on the ELISA assay of serum IgM antibody to RV. The detection kit is almost entirely dependent on imports, which is very expensive, and is difficult for routine clinical laboratory to carry out. Anti-RV antibody test results depend on the specificity of the antigen preparation, which determines the accuracy of test results. The main three RV structural proteins - the envelope glycoprotein E1, E2 and capsid protein C have higher immunogenicity in the human body, and can induce a strong antibody response. Among them, E1 includes the majority of RV area and hemagglutination inhibition immune epitope, and studies showed that the degree of sequence variation of E1 gene is similar to that of whole-plant. The biological role of E2 protein is not clear, and capsid protein C is mainly related with viral replication. Due to the existence of cross-antibody responses, they can't be used as a diagnostic antigen. Therefore, among the three kinds of structural proteins, E1 protein is the most suitable genetic engineering autoimmune antigen, and can be used as diagnostic reagents and vaccines.In this study, we extracted the RV genomic RNA from the measles, mumps and rubella triple vaccine, and reversed it to cDNA with transcriptase. Its genome was preservated and amplificated the target gene. Then, through genetic engineering methods, E1 gene conservative, the N-terminal 196-305 amino acid peptide E1-N with high immunogenicity was expressed. After the E1-N gene fragment applified using PCR was inserted into the prokaryotic expression vector pGEX-2T plasmid, the E1-N recombinant plasmid pGEX-2T/E1-N was constructed. The recombinant plasmid pGEX-2T/E1-N, confirmed by digestion with restriction endonuclease EcoR I, BamH I and sequencing, was further transferred into E.coli BL21. Fusion protein was induced to express with isopropyl-β-D thiogalactoside (IPTG) at 16℃and 37℃respectively. As a result, glutathione-S-transferase (GST) and the E1-N fusion protein were expressed mainly in the supernatant at 16℃, while the fusion protein mainly in inclusion body at 37℃.Through the above-mentioned study, the E1 envelope glycoprotein-specific gene sequence was amplified and confirmed by sequencing, which had the same gene sequence with GeneBank reported. With IPTG-induction, recombinant fusion peptide E1-N was expressed. Results showed that the best concentration of IPTG is 1 mmol/L, the best time is 5 h, and the best temperature is 16℃. To avoid the formation of inclusion bodies, recombinant fusion protein was expressed in soluble form in the supernatant by low-temperature-induced, which avoided the recombinant fusion protein denaturation and renaturation process, thus greatly simplified the operation. Therefore, the recombinant fusion protein expression is the basis of further the development of RV-specific Detection Kit and development of new genetically engineered vaccine.
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