Cloning, Expression And Product Purification Of HSV Glycoprotein G Gene Fragment And Preliminary Application Of The Detection Reagents For HSV Antibody

Herpes simplex virus (HSV) can be divided into two serotypes, types 1 (HSV-1) and 2 (HSV-2). The infectious rate of HSV is high both in developing countries and developed countries. Herpes simplex encephalitis (HSE) is a rare but very serious disorder caused by herpes simplex virus type 1 and leads a bad prognosis, fetus malformation and genital inflammation are associated with herpes simplex virus type 2. Early antiviral therapy of HSV infections had a good effect. Therefore an early, rapid, specific and sensitive diagnostic technique is essential. Now we usually determine herpes simplex virus infections by immunoassay, the detection of type-specific IgM antibodies in sera from HSV infected individuals can be used for early diagnosis of acute infection. Glycoprotein G is the major target of humoral immune response to HSV and can be as a standard antigen for type-specific serological diagnosis of herpes simplex virus infections.Section One: Cloning and High Expression of Herpes Simplex Virus Type 1 Glycoprotein G in Escherichia ColiA herpes simplex virus type 1 glycoprotein G gene fragment containing dominant antigenic determinants confirmed by analysing amino acid sequences was cloned by PCR and inserted into plasmid vector pGEX-4T-2 digested with BamU I and EcoR I, then the recombinant plasmid was transformed into E.coli TGI. The sequence of gG-1 gene obtained was identified by PCR and sequenceing.The recombinant was induced by IPTG for expression. SDS-PAGE analysis indicated that a chimeric protein of HSV-1 glycoprotein G fragment with GST was highly expressed in E.coli and accounted forabout 30% of total bacterium protein. Relative molecular weight of the expressed HSVl-gG/GST chimeric protein was about 43.5kD.The engineering E.coli for the high-level expression of herpes simplex virus gG-1 gene was established successfully. This lays a foundation of purification of the recombinant antigen with good antigenicity and specificity.Section Two: Purification and Preliminary Identification of the Expressed HSVl-gG ProteinThe recombinant strain expressing HSVl-gG protein by IPTG induction was lyzed by ultrasonic wave and harvested by centrifugation. The expressed HSV-1 glycoprotein G mainly existed in the form of soluble protein. Then the supernatant was purified by DEAE-Sepharose FF anion exchange column chromatography following ammonium sulfate fractionation step, and then detected by SDS-PAGE. The target protein eluted with 200mmol/L NaCl elution buffer was purified by DEAE-Sepharose FF anion exchange column again. The purified gG-l/GST chimeric protein existed in 150mmol/L NaCl elution buffer, and its purity is about 90%.Using the purified protein as antigen, we tested anti-HSVl IgM/IgG positive sera and normal sera by indirect enzyme-linked immunnosorbent assay (ELISA) and western blot. The results indicated the expressed gG-l/GST protein was reactive to positive serum and non-reactive to normal serum in ELISA and western blot. All above results preliminarily confirmed the chimeric protein could be produced and purified easily, and the protein has strong antigenic determinants.We successfully cloned and expressed the gG-1 recombinant protein antigen, and it would be very useful for developing the reagents for immunological diagnosis of HSV-1 infections.Section Three: Preliminary Development and Application of the Detection Reagents for HSV-1 AntibodyThe purified recombinant protein (HSVl-gG/GST) was coated onto microtiter plates as antigen. The secondary antibodies were horse radish peroxidase (HRP)-conjugated goat anti-human IgG/IgM. 46 sera from clinical random patients, 20 anti-HSVl IgM positive sera and 51 normal sera were tested by indirect ELISA for detecting anti-HSVl IgG/IgM antibodies. The results indicated that the recombinant antigen has a specific reaction with about 72% of 46 random sera (IgG) and 20 anti-HSVl IgM positive sera (IgM), whereas no reaction with 51 sera (IgM) from normal persons. All above findings showed the chimeric protein has good ant...