ABSTRACT
Background: Biosynthetic teriparatide [1-34] [TPD] is a N-terminally truncated version of human parathyroid hormone [hPTH]. The recombinant form of this polypeptide has been expressed in Escherichia coli [E. coli] and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis [B. subtilis] was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities
Methods: A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5' to 3', was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into B. subtilis. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG
Results: The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium
Conclusion: TPD may be successfully expressed and secreted in B. subtilis; however, optimization of expression conditions is required for enhancing target protein yield
ABSTRACT
Despite availability of an effective vaccine against hepatitis B virus [HBV], the global prevalence of this virus infection has not diminished significantly. Contrary to numerous other human viruses, HBV does not have the ability to propagate in cell culture. However, infectious virus has been produced by transfection of human hepatoma cells with plasmids that contain full length HBV genome. Generation and optimization of appropriate cell culture systems can help us in demonstrating the quality of genome replication by PCR as well as expression of surface antigen secretion. Interferon stimulating genes [ISGs] are usually produced in response to interferon and can be determined as a measure of response to IFN-therapy. Therefore, in pharmacological studies, in addition to assessing the effects of a medicine on viral determinants of replication, its' effects on stimulation of various ISGs, as indicators of innate immune responses, can be achieved. In this study, we transfected the Huh-7 hepatoma cell line with pCH-9/3091. HBsAg production and viral mRNA transcription were subsequently evaluated. In this system, by using ISGs-specific primers, the ISG mRNAs recognition method was optimized and utilized. Huh-7 cells supported HBV replication. The peak HBsAg secretion was observed at 72 h post-transfection. By using designed primers for the S and pg/pC regions, transcription and genome replication of the virus was shown. RT-PCR results for ISG production by transfected cells showed no role for HBV in enhancement of ISGs levels in Huh-7 cells. The results indicated that this system can be used for functional studies of HBV-specific genes as well as assessment of the effects of new drugs or new vaccines. In addition, it may be used to study the mechanisms of drug resistance that have resulted in difficulties in response to HBV antivirals, including IFN-alpha
ABSTRACT
Hepatitis C virus [HCV] envelope glycoprotein-2 [E2] inhibits the interferon [IFN]induced, double stranded RNA activated protein kinase [PKR] via PKR eukaryotic initiation factor-2a phosphorylation homology domain [PePHD]. Present study examined the genetic variability of the PePHD in patients receiving interferon therapy. The PePHD region from HCV genotype 1a/1b infected patients receiving IFN was amplified by reverse transcriptase polymerase chain reaction [RT-PCR] and analyzed using bidirectionaly sequencing. The PePHD sequence was different in pretreatment isolates from three months treated patients. It was shown that the major PePHD quasispecies could change after three months IFN therapy and in one patient; the major PePHD quasispecies could change after six months IFN therapy. These mutations were occurred at codons 665, 666 and 667 of followed-up samples and at codons 660, 661, 666 and 670 of randomly treated patients. Some of these mutations were similar to those reported in previous studies. Other mutations were also detected in upstream and downstream regions of PePHD which may have influenced the structure, conformation and configuration of this region and thereby suppressing PePHD inhibitory properties. In conclusion our data suggested that HCV E2 PePHD may play an important role in determining the interferon response among Iranian HCV infected patients
Subject(s)
Humans , Male , Female , HEPACIVIRUS-CLASSIFICATION , Treatment Refusal , Polymerase Chain Reaction/methods , eIF-2 Kinase , Genetic VariationABSTRACT
Hepatitis Delta virus [HDV] is a degenerate RNA virus or virusoid and a satellite of Hepatitis B virus [HBV]. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, an RT-nested PCR method was set up to detect delta infection from serum samples. Moreover, the target amplified sequences corresponding to the Hepatitis delta antigen [HDAg] C-termini were used for genotyping. The results showed that 63.6% [23 of 36] of [HDAb] positive serum samples [as determined by ELISA] were also positive for HDV-RNA. Sequencing and phylogenic analysis of three Iranian HDV isolates revealed the most homology [93%] with an Italian isolate indicating a close relationship and probably a common origin for these isolates