A Sensitive and Rapid Assay for Investigating Vertical Transmission of Hepatitis B Virus via Male Germ Line Using EGFP Vector as Reporter

Abstract

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed.