Anti-His tag antibody [FITC]

Due to the aquatic animal pathogens are numerous and specific, the pathogen invasion mechanisms are more complicated. The cell surface receptors play vital roles to understand these mechanisms. Syndecan is a cell surface protein and could function as a receptor involved bacteria and virus infections. But there are few studies on the function of syndecan in shrimp and their interaction with aquatic bacterial pathogens. In the present study, we identified a syndecan receptor gene from Macrobrachium rosenbergii and analyzed its functions during the bacterial infections. The MrSDC was expressed in various tissues and presented a constitutive expression distribution except in eyestalk. Recombinant MrSDC-his tag protein was expressed in the E. con BL21 with pET30a/MrSDC plasmid and exhibited a broad bacterial binding activities. The inhibition of MrSDC expression by dsRNA interference and antibody blocked could significantly reduce the number of Aeromonas hydrophila in hepatopancreas compared with the control. The overexpression of MrSDC by mRNA injection could significantly increase the number of A. hydrophila. In addition, the functional role of syndecan heparan sulfate chains in bacterial recognition was also studied. After extra injection of heparan sulfate in vivo, the bacterial numbers and accumulative mortality of M. rosenbergii were significantly higher than control groups and exhibit a dose effect. All these data could indicate that the cell surface syndecan protein could function as mediator in bacterial infections by the heparan sulfate chains. Our present study will provide new insights into the functions of shrimp syndecan.

Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 degrees C. Moreover, it was active over the broad temperature region of 30-60 degrees C, and stable within the pH range of 4.5-10.0. In addition, Co2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low- concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the beta-1,3-1,4 glucan linkage from barley. The Michaelis-Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 mu mol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.