Prokaryotic Expression And Drug Resistance Detection Of HBV Polymerase RT Region Containing ADV-Resistant Mutations

Background Hepatitis B virus (HBV) is a hepatotropic DNA virusthat replicates by reverse transcription. HBV infection is still one of themost serious and prevent heath problems, accounting annually for1milliondeaths worldwide from cirrhosis, liver failure, and hepatocellularcarcinoma. Adefovir dipivoxil (ADV), one of current nucleostide analogsdrug, could inhibit HBV replication by target HBV polymerase activity.But the suppression is insufficient to blockHBV replication, and long termuse may lead to drug resistance, resulting treatment failure.The polymerase (pol) protein consists of four domains: terminalprotein (TP), spacer domain, pol/reverse transcriptase (RT), and RNase H(RH). The polymerase protein participates directly in the HBV genomereplication, which contains DNA polymerase and RNAseH activity.Lacking of error correction capabilities for HBV DNA polymerase, itgenerates a high rate of mutations during the replication process. This would decline the affinity between drugs and HBV polymerase. Manyreports have confirmed that rtN236T and rtA181V/T mutations, at loci inthe reverse-transcriptase (RT) domain of HBV polymerase, are associatedwith ADV failure. Clinical studies have shown that treatment during orafter treatment in patients with elevated alanine aminotransferase activityoccurs all of a sudden, viral load increased again, the previous drugs cannot inhibit replication of the virus. The sensitive detection of suchmutations before or early in treatment could assist in optimizing antiviraltreatment. Therefore, developing a sensitive, specific, accurate, and easy toperform method for the detection of ADV-resistant HBV is of great clinicalimportancy.Objects1: Expressing the Addfovir-resistant mutant of reversetranscriptase (RT) domain of HBV polymerase in prokaryotic cell in orderto investigate the inhibition mechnism of ADV target for RT domain.2: Todevelope a sensitive, specific, accurate, and easy to perform method for thedetection of ADV-resistant HBV.Methods1: Hepatitis B virus (HBV) reverse transcriptase (RT domain)was cloned into vector pHis-SUMO, and the plasmid with Adefovir-resistant (181T/V+236T) motifs were obtained by site-directed mutagenesis.The recombinant plasmid SUMO-RTWT、SUMO-MT1and SUMO-MT2were transformed into Rosetta (DE3), and the expressions products weredetected by SDS-PAGE and Western blotting. The condition for expression was optimized, and the structure of RT domain of HBVpolymerase was modeled.2: The specific peptide nucleic acid probes weredesigned for the ADV resistance characteristic and immobilized to themembrane via an amino linker. Then we conducted a series of experimentsto optimize the probe concentration, hybridization conditions, elutionconditions in order to set up the best reaction conditions which could beemployed for detecting the ADV resistance characteristic of HBV. Finally,the diagnostic reliability of this assay (specifically and sensitively) wasevaluated in clinal samples.Results We succeeded in cloning and expression of three kinds offusion protein, purifying one of them. Very unstable due to less protein andprotein crystals could not be completed. Adopting standard plasmid lines tocomplete the reverse hybrid optimization and peptide nucleic acid detection,testing for follow-up tests on clinical samples.