Cloning And Expression Of HSV-Ⅰ Glycoprotein G Gene Fragment And Preparation Of Monoclonal Antibody Against The Expressed Protein

Herpes simplex virus, including HSV-â… and HSV-â…¡two serotypes, is a representative and typical herpes virus of the Herpesviridae. HSV-â… is the most common cause of sporadic encephalitis and keratitis. Complications from HSV-â… infection may be severe in the immunocompromised host (pregnant women) and neonate. Since the lack of effective prophylactic measures, early and definitive diagnosis of infected pregnant women is particularly important to stop the vertical transmissions. At present, detection of HSV-â… antibody is the most commonly used method of HSV detection.In order to enhance the sensitivity, specificity and accuracy of HSV-â… antibody detection, we intend to express the recombinant gGâ… protein and develop its McAb, which could be a basis for the development of ELISA assay kit and promote the further development of HSV-â… type-specific serological detection. Around this purpose, we conducted the following research work.1. Cloning of gGâ… gene fragment and construction of recombinant plasmidAccording to the homology sequence of gGâ… about different HSV-â… train, we designed a pair of primers, then the gene fragment was amplified by PCR using the primers, the production contains the corresponding gene fragments of the amino acid sequence about dominant antigenic determinants (No.112-127 aa). The PCR production and plasmid vector pGEX-4T-1 were digested with EcoR I and Sal I, then inserte PCR production into plasmid vector, constructe the recombinant plasmid, and identificate it. Finally, we obtained the recombinant plasmid containing highly conserved target gene fragment.2. Purification and identification of the expressed GST/gGâ… ProteinThe pGEX-4T-1/gGâ… was transformed into BL21, then induced by IPTG for expression, the expressed production was identificated by SDS-PAGE. The recombinant strain was lyzed by ultrasonic wave and harvested by centrifugation. Then the supernatant was purified by affinity chromatography, the character of GST/gGâ… protein was identificated by indirect ELISA. Finally, we obtained the recombinant protein that remaining the antigenicity and specificity of native protein.3. Preparation of McAb against recombinant protein about gGâ… After strictly and regularly immunized, cell fusion was carried out using the appropriate BALB/c mice, hybridoma was screened at the appropriate time. Unfortunately, we did not obtain hybridoma of specific secretory antibody response to gGâ… , but we obtain two hybridoma of the GST-specific antibody secretion. So, we thoroughly analyzed the causes of test failures. In order to eventually obtain the specific monoclonal antibody for gGâ… recombinant protein, we had proposed to improve the program.