Expression and Purification of Neurotoxin-Associated Protein HA-33/A from Clostridium botulinum and Evaluation of Its Antigenicity

Background: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Methods: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. Results: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. Conclusion: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice. Key Words: Botulinum neurotoxin, Expression, Purification     

Bending moment resistance of dowel corner joints in case-type furniture under diagonal compression load

We investigated bending moment resistance under diagonal compression load of corner doweled joints with plywood members. Joint members were made of 11-ply hardwood plywood of 19 mm thickness. Dowels were fabricated of Beech and Hornbeam species. Their diameters(6, 8 and 10 mm) and depths of penetration(9, 13 and 17 mm) in joint members were chosen variables in our experiment. By increasing the connector's diameter from 6 to 8 mm, the bending moment resistance under diagonal compressive load was increased, while it decreased when the diameter was increased from 8 to 10 mm. The bending moment resistance under diagonal compressive load was increased by increasing the dowel's depth of penetration. Joints made with dowels of Beech had higher resistance than dowels of Hornbeam. Highest resisting moment(45.18 N·m) was recorded for joints assembled with 8 mm Beech dowels penetrating 17 mm into joint members Lowest resisting moment(13.35 N·m) was recorded for joints assembled with 6 mm Hornbeam dowels and penetrating 9 mm into joint members.     

Analysis of factors affecting diagonal tension and compression capacity of corner joints in furniture frames fabricated with dovetail key

This study was conducted to analyze the effect of joint type, and numbers and types of dovetail keys on diagonal tension and compression performance of corner joints in a furniture frame. Joint members were cut from white fir lumber. Butted and mitered joints were constructed with one and two dovetail key(s) with butterfly and H shapes. Joints were glued by polyvinyl acetate (PVAc) and cynoacrylate (CA). Compression capacity of joints was higher than diagonal tension. Mitered joints were stronger than butted ones. Butterfly dovetail keys were superior to H shape keys. Double keys performed better than single key. Experimental joints glued with PVAc were stronger than those glued with CA glue and control specimens. In terms of strength, butterfly dovetailed joints were comparable with doweled joints.     

Comparative Study of Immunological and Structural Properties of Two Recombinant Vaccine Candidates against Botulinum Neurotoxin Type E

Background

Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains 93 amino acid residues from recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). Both proteins have an identical region (48 aa) that contains one of the most important BoNT/E epitopes (YLTHMRD sequence).

Methods

The recombinant protein efficiency in antibody production, their structural differences, and their BoNT/E-epitope location were compared by using ELISA, circular dichroism, computational modeling, and hydrophobicity predictions.

Results

Immunological studies indicated that the antibody yield against rBoNT/E-HCC was higher than chimer protein. Cross ELISA confirmed that the antibodies against the chimer protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups (anti-chimer and anti-rBoNT/E-HCC antibodies) were able to recognize other proteins. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC.

Conclusion

The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the chimer protein, which is strongly in agreement with immunological results. Iran. Biomed. Key Words: Botulinum neurotoxin type E (BoNT/E), Cross ELISA, circular dichroism, Computational modeling, recombinant vaccine-candidates     

Bending moment resistance of corner joints constructed with spline under diagonal tension and compression

We determined the effects of the penetration depth and spline material and composite material type as well as joining method on bending moment resistance under diagonal compression and tension in common wood panel structures. Composite materials were laminated medium density fiberboard (MDF) and particle board. Joining methods were buttand miter types. Spline materials were high density fiber board (HDF).The penetration depths of plywood, wood (Carpinus betolus) and splinewere 8, 11 and 14 mm. The results showed that in both diagonal compression and tension, MDF joints are stronger than particle board joints,and the bending moment resistance under compression is higher compared with that in tension. The highest bending moment resistance under tension was shown in MDF, butt joined using plywood spline with 8 mm penetration depth, whereas under compression bending moment resistance was seen in MDF, miter joined with the HDF spline of 14 mm penetration depth.