A Study On The Mutations Of Basal Core Promoter And Precore Gene Of Hepatitis B Virus Genome In E Antigen-Negative Sera

Hepatitis B virus (HBV) is the prototype member of the hepadnavirus family. It has a circular and partially double-stranded DNA genome of about 3.2 kb containing four overlapping open reading frames: the C region, S region, P region and X region. The whole HBV particle is Dane particle, and it is a 42 nm global particle. HBV is known as the minimal DNA virus that is harmful to human at present. The high efficiency of the genetic information of HBV DNA is rare in the nature world. The replication of HBV DNA passes through reverse transcription. As the reverse transcriptase is lack of effective function of proofreading the bases, the HBV genome is easier to mutate than other DNA viruses. It will bring new problems to the preventability, diagnosis, remedy and prognosis of the diseases.In this experiment, we meliorated the way of seething with disruption liquid and took the way to extract HBV DNA templates from the serum samples. Compared with another two ways, this one was quicker, simpler and more efficient. Consulting the HBV genome in GenBank , We designed three specific primers . The products of PCR include BCP(basal core promoter) and PreC gene. In the 16 samples of HBV DNA positive sera, 13 samples were negative and 3 samples were positive for HBeAg. The samples were all amplified by heminested-PCR. After purified and recovered, the products were cloned to pUCm-T vectors. The pUCm-HBV-403 recombinations were filtrated by α -complement and identified by PCR and restriction endonuclease. According to sequence and aligning HBV BCP and PreC gene, we found the mutations of BCP were grouped in TA1 TA2 and TA3 of TATA-like boxes. There were no insertion or deletion mutations, which were different from the report of Dong jing in Beijing. TA4 was very conservative, and there was no mutation. One of the hot mutations in BCP was at nucleotide 1799 position (C→G), that was synonymous mutation to X protein. The mutations in PreC gene were grouped at nucleotide 1896position (G—A), which rendered the progression. Furthermore, in this research a novel insertion mutation was found in DR1. DR1 and DR2 play an important role in the replication and annulus.Mutations in BCP and PreC gene can cause e antigen seroconversion and escape from the recognition by the host's immune system. Some of the mutations will lead to serious liver damages, and some will affect the curative effect of interferon. In order to improve the level of diagnosis and remedy of HBV, we should attach more importance to the research of HBV BCP and PreC gene mutations.