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Prokaryotic Expression of Specific Fragment of Rubella Virus E1 Gene

Author: CaoJing
Tutor: HuangYuFeng;LuJinChun
School: Nanjing Medical University
Course: Immunology
Keywords: Rubella virus E1 - specific peptides Recombinant plasmid The prokaryotic expression
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 23
Quote: 0
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Rubella virus (rubellavirus, RV), also known as German measles virus, is the only member of the genus Togavirus rubella virus antigen no cross with other viruses in the the Togavirus Branch. RV TORCH syndrome pathogens (cytomegalovirus, herpes simplex virus, rubella virus, Toxoplasma gondii) one, it is important to fetal teratogenic factor, can pass through the placenta and infect the fetus, resulting in newborns suffer from congenital rubella syndrome (congenitalrubellasyndrome , CRS), can be expressed as cataracts, deafness, heart disease or mental developmental disorders. Infection in pregnant women the RV sooner, the greater the probability of newborns suffering from CRS. RV is also the impact factor of male infertility, male reproductive system infection of TORCH, can cause orchitis, epididymitis, prostatitis, and male infertility. Currently, immunological detection means has been widely used in RV infection clinical diagnostic methods, such as the hemagglutination inhibition test (HIA), specific antibody IgM, IgG and RV protein antigen detection. The RV infection screening is mainly based on the ELISA assay specific serum IgM anti-RV antibody test kit is almost entirely dependent on imports, the price is expensive, it is difficult to carry out suitable for clinical routine laboratory. The accuracy depends on the detection results of anti-RV antibodies the corresponding antigen preparation, therefore, the specificity of the antigen determines the accuracy of test results. Although the RV three structural proteins - the envelope glycoprotein E1, E2 and capsid protein C have a higher immunogenicity in the human body, can induce a strong antibody response. Including the most hemagglutination inhibition RV area and immune epitopes which E1, and studies have shown that a similar level of the degree of variation between the E1 gene sequence of strain and the whole plant sequence variation the E2 protein biological role is not clear, the capsid protein C viral replication, the latter due to the presence of cross antibody response specific antigen as a diagnostic. Therefore, the the RV three kinds of structural proteins, E1 protein is most suited as a genetic engineering antigen used immunological diagnostic reagents and vaccine. This study from the measles, mumps and rubella attenuated live vaccine RV genomic RNA extracted, reverse transcribed into cDNA, to facilitate the preservation and further amplified gene in its genome. Then through genetic engineering techniques to express the E1 gene conservative, high immunogenicity of 196-305 amino acids of the amino-terminal peptides of E1-N. Gene encoding E1-N fragment was amplified by PCR, and inserted into the prokaryotic expression vector pGEX-2T plasmid construct expression E1-N recombinant plasmid pGEX-2T/E1-N of digestion and sequencing confirmed the plasmid transformation E.coliBL21 strains, induced by isopropyl-β-D thiogalactoside (IPTG) to induce expression of fusion protein, respectively, at 16 ° C and 37 ℃ results: 16 ℃ induced expression The fusion protein of glutathione-S-transferase (GST), and E1-N are mainly present in the supernatant; to 37 ℃ induced fusion protein is mainly present in the inclusion bodies. The obtained through glutathione agarose bead affinity chromatography purified GST/E1-N fusion protein. Through the above study, we amplified the envelope glycoprotein E1-specific gene sequences were confirmed by sequencing the gene sequence with GenBank reported consistent induction with IPTG Recombinant expression plasmid pGEX-2T/E1-N expression of recombinant fusion peptides The optimal concentration of IPTG 1mmol / L, the optimal induction time of 5h optimal induction temperature of 16 ° C. Induced by low temperature to avoid formation of inclusion bodies, the recombinant fusion protein expressed in soluble form in the supernatant, thus avoiding the recombinant fusion protein denaturation and refolding process, greatly simplifying the operation. Therefore, in this study the expression of E1-specific protein for the further development of specific the RV detection kits and development of new genetically engineered vaccine provides an experimental basis and methodological basis.

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