Hepatitis B is a major public health problem worldwide, which may lead to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The hepatitis B core antigen (HBcAg) is one of the major viral proteins, which forms the inner core of hepatitis B virus (HBV) particles. In this study, filamentous bacteriophage M13 was genetically modified to display the polypeptides of HBcAg in order to develop an alternative carrier system. HBcAg gene was inserted into the minor coat protein (pIII) gene of M13, and HBcAg was expressed on the phage surface as a whole protein. Antigenicity and immunogenicity of HBcAg were tested by immunizing BALB/c mice three times with HBcAg-displaying recombinant phages. After successful immunization, one of the mice with high antibody titer to HBcAg was selected for fusion, and four monoclonal antibodies specific for HBcAg were developed. This result showed that HBcAg-displaying recombinant bacteriophages are immunogenic and can potentially be used for the development of monoclonal antibodies.
Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.
The horse has been a food source, but more importantly, it has been a means for transport. Its domestication was one of the crucial steps in the history of human civilization. Despite the archaeological and molecular studies carried out on the history of horse domestication, which would contribute to conservation of the breeds, the details of the domestication of horses still remain to be resolved. We employed 21 microsatellite loci and mitochondrial control region partial sequences to analyse genetic variability within and among four Anatolian native horse breeds, Ayvacık Pony, Malakan Horse, Hınıs Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: first, to produce data from the prehistorically and historically important land bridge, Anatolia, in order to assess its role in horse domestication and second, to analyse the data from a conservation perspective to help the ministry improve conservation and management strategies regarding native horse breeds. Even though the microsatellite data revealed a high allelic diversity, 98% of the genetic variation partitioned within groups. Genetic structure did not correlate with a breed or geographic origin. High diversity was also detected in mtDNA control region sequence analysis. Frequencies of two haplogroups (HC and HF) revealed a cline between Asia and Europe, suggesting Anatolia as a probable connection route between the two continents. This first detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolia's role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey.
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