A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA–CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA–CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA–CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood.

Recombinant fusion protein by lysozyme and antibacterial peptide enhances ischemic wound healing via angiogenesis and reduction of inflammation in diabetic db/db mice

Background & aims Lysozyme and antibacterial peptides have been reported to broad-spectrum antibacterial activity and can further improve wound healing. The aim of this study was to assess the effectiveness of a recombinant fusion protein created by combining lysozyme and an antibacterial peptide in forming new vessels and wound healing in an ischemic hind limb. Methods An ischemic hind limb model was established by isolation and ligation of the femoral artery in diabetic db/db mice. Cutaneous wounds were created with or without ischemia. Adductor muscles and wounds were treated with or without the fusion protein. Results The fusion protein accelerated ischemic diabetic wound healing and attenuated impairment of ischemic adductor muscle . Further, the fusion protein elevated expression of platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) protein and mRNA in ischemic adductor muscle, reduced levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in serum and expression of phosphorylated nuclear factor κB (p-NF-κB) and p-IKB α in ischemic adductor. The fusion protein also enhanced levels of phosphorylated VEGF and PDGF receptors in the ischemic adductor muscles from diabetic db/db mice. Conclusion The data showed that the beneficial effects of the fusion protein on ischemic wound healing may be associated with angiogenesis and reduction of inflammatory response in the ischemic adductor muscles of diabetic db/db mice.

Induration or erythema diameter not less than 5 mm as results of recombinant fusion protein ESAT6-CFP10 skin test for detecting M. tuberculosis infection

Background Recombinant fusion protein ESAT6-CFP10 (EC) is a newly developed skin test reagent for detecting Mycobacterium tuberculosis (M. tuberculosis) infection. In this study, we evaluated whether induration and erythema could be used as diagnostic indicators for EC skin test to detect M. tuberculosis infection. Methods A total of 743 tuberculosis patients and 1514 healthy volunteers underwent an EC skin test. The diameters of induration and erythema were measured with Vernier caliper, 24 h, 48 h, and 72 h after skin testing. Related indicators of EC reagent diagnostic test were tested, and the diagnostic effects of the four diagnostic indicators for EC skin test were compared. Results The sensitivity of induration / erythema measurement was lower at 24 h after EC skin test than at 48 h or 72 h (P<0.01). There was no difference in consistency (P = 0.16) between induration with clinical diagnosis, and erythema with clinical diagnosis at 48 h (88.88 and 90.16%, Kappa value was 0.75 and 0.78, respectively). In patients, the sensitivity of erythema measurement was higher than induration measurement (P<0.01). In healthy volunteers, the specificity of erythema measurement was lower than induration at 24 h after skin test, but there was no difference at 48 h after skin test (P = 0.22). In BCG vaccination volunteers, the specificity of induration and erythema were higher than 90%. In addition, there was a high consistency of induration and erythema. When induration or erythema was used as a positive diagnostic indicator, the sensitivity of the EC skin test was improved, and was no different from the other three indicators in terms of specificity and consistency with clinical diagnosis. Conclusions Induration or erythema diameter not less than 5 mm could be used as a diagnostic indicator for detecting M. tuberculosis infection. Trial registration Phase III clinical trial of recombinant Mycobacterium tuberculosis ESAT6-CFP10 allergen; CTR20150695; registered in December 16, 2015.

Expression of Recombinant Fusion Protein from Local Isolate of Newcastle Disease Virus and Antibody Response to Recombinant Fusion Protein in Broiler Chickens Post-Vaccination

This research aimed to express and purify the recombinant Fusion (F) protein of Newcastle Disease Virus (NDV) from a local isolate in Galur, Kulon Progo, Indonesia (0663/04/2013) from recombinant vector plasmid pBT7-N-His F, and to study the antibody response in the broiler sera which were injected with pure recombinant F protein compared with treated broilers that were vaccinated with commercial inactive NDV vaccines and control broilers without vaccination. The results showed that the recombinant F protein of NDV was successfully expressed, purified and visualized by SDS-PAGE with Coomassie Brilliant Blue staining and Westernblotting methods as a specific recombinant F protein with a molecular weight of 28 kDa. The pure recombinant F protein then was injected into broilers to determine the antibody response in broiler serum. Indirect ELISA showed that the production of antibodies was high in F protein vaccinated groups in comparison with other treated and control groups. The recombinant F protein has potential to be developed as a recombinant vaccine candidate after truncating the 6x His-tag part to obtain higher antibody respond if compared with antibody production in broiler serum post vaccinated with some commercially available broiler vaccines.