The results indicated that this detection effect of the P20 antibody was very significant and that it exhibited optimal specificity for the four genotypes of HBV pol (Figure 1D). Open Tiaprofenic acid in a separate window Figure 1. Characterization of the mAb specific for HBV pol (A). results indicated significant antiviral effects caused by these two antibodies especially P12. In summary, the present study established an antibody which was denoted P20. This antibody can be used to detect HBV pol expression by four HBV genomes via Tiaprofenic acid WB analysis. In addition, the antibody denoted P12 could exert antiviral effects via intracellular expression, which may provide a encouraging approach for the treatment of chronic hepatitis B. KEYWORDS: HBV, polymerase, antibody, detection, therapy Introduction HBV can constantly replicate and infect liver cells and is considered an important pathogenic factor that seriously endangers human health [1,2]. Multiple intracellular viral proteins, such as HBx, HBcAg and HBV polymerase, are critical for HBV replication. HBV pol is one of the HBV intracellular proteins involved in almost the entire HBV viral replication process [3,4]. This enzyme is essential for the life cycle of the computer virus and regulates several processes including viral RNA binding, RNA packaging, protein initiation, template conversion, DNA synthesis and RNA degradation [5]. Nucleos(t)ide analogs (NAs) can take action around the polymerase domain name and inhibit its activity, resulting in decreased HBV DNA levels [6]. In addition, several recent studies have reported that HBV polymerase interacts directly with STING and RIG-I, thereby interfering with the Tiaprofenic acid replication of viral DNA and RNA through pattern acknowledgement receptors in the host immune system [7C9]. HBV pol ultimately reduces the type I interferon response [10C13]. These investigations may provide potential evidence for any therapeutic approach for HBV. Therefore, it is important to study the role of HBV pol in chronic HBV contamination. However, the current research field lacks specific and high-quality antibodies for detecting HBV pol, which may hinder further research. In the present study, the HBV pol (159C406 aa) protein was used as a target for the development of specific monoclonal antibodies that recognize native HBV pol NFIL3 and the antiviral effects Tiaprofenic acid of the selected antibodies were evaluated. Materials and methods Plasmid construction The B-type HBV1.3 ploidy was used as a template, based on the full-length sequence of the genotype B gene (“type”:”entrez-nucleotide”,”attrs”:”text”:”GU357842″,”term_id”:”284927734″,”term_text”:”GU357842″GU357842) obtained from Genbank. The primers were designed to amplify the (159C406) DNA fragment. The sequence of the forward primer with a BamHI restriction site at the 5? end was the following: 5?- TGCACCACCACCACCACCACGGATCCATGAAAAGAGAGTCCACACGTAG ?3? and that of the reverse primer with an EcoRI restriction site at the 5? end was as follows: 5?- CAAGCTTGTCGACGGAGCTCGAATTCTTATTTTGGCCAAGACACACGGGTGTTC-3?. A 6-His tag series was added to the beginning of the (159C406) and was ligated to the pTO-T7 plasmid vector. Protein expression and purification The 6His-HBV pol (159C406 aa) protein was expressed following the induction of IPTG and the formation of the inclusion body in plasmid, which expressed the full length gene. Immunofluorescence and WB analysis were used to detect the binding activity of the antibody to the full-length HBV pol. The 2 2?C8 antibody was used as a control. Immunofluorescence assays exhibited that this P3, P5 and P20 antibodies indicated binding affinity to the full-length HBV pol expressed by the eukaryotic system (Physique 1B). However, P12 was not reactive (data not shown). Both P3 and P20 antibodies indicated WB reactivity with the full-length HBV pol. Notably P3 exhibited optimal specificity. The P3 antibody exhibited an extra band compared with that of the 2C8 antibody, which may have been caused by the epitope difference between these two antibodies (Physique 1C). The P3 antibody also exhibited a band at 250C300 (estimated) kDa size in all samples tested (Physique 1C). We considered this to be a nonspecific band because the same band was present in all lanes, including the control. The findings may provide a tool for studying the different functions of HBV pol. Even though specificity of the P20 antibody was poor, it exhibited an ideal detection effect on HBV pol, which was expressed in cells transfected with the HBV genome (Physique 1C). Previous studies have not reported an antibody with comparable affinity as that in the current study. To confirm our results, we used four genotypes (A, B, C, D) of HBV 1.3 ploidy plasmid-transfected cells and two HBV cell lines (HepG2.2.15 and HepAD38) to verify the detection.