The celF gene from the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes encodes a 118.3-kDa cellulose-binding endoglucanase, endoglucanase F (EGF). This enzyme possesses an N-terminal cellulose-binding domain and a C-terminal catalytic domain. The purified catalytic domain displayed an activity profile typical of an endoglucanase, with high catalytic activity on carboxymethyl cellulose and barley -glucan. Immunoblotting of EGF and the formerly characterized endoglucanase 2 (EG2) from F. succinogenes with antibodies prepared against each of the enzymes demonstrated that EGF and EG2 contain cross-reactive epitopes. This data in conjunction with evidence that the proteins are the same size, share a 19-residue internal amino acid sequence, possess similar catalytic properties, and both bind to cellulose allows the conclusion that celF codes for EG2.
Antibodies against Pseudomonas aeruginosa LPS are generally protective, however, this protection is usually serotype-specific. Thus the generation of antibodies against the more conserved core-lipid A epitopes has a potential for being more broadly cross reactive. Our laboratory has previously produced several monoclonal antibodies (mAb) against the core region and lipid A region of P. aeruginosa LPS. In this study, we cloned the immunoglobulin genes from mAb 7-4, an antibody with specificity for the inner core region of P . aeruginosa LPS. VH and VL genes of 7-4 were cloned into both of the M13-derived phagemid vectors, pComb3 and pComb8. 14 pComb3/VH/VL and 6 pComb8/V H /V L recombinant clones were isolated. The presence of recombinant F(ab) molecules in the periplasmic extracts of Escherichia coli was confirmed by Western immunoblots of these extracts with goat anti-mouse F(ab')2 and goat anti-mouse kappa light chain antibodies under non-reduced conditions. Recombinant 7-4 antibody also interacted with LPS prepared from a rough mutant P. aeruginosa strain AK43 in Western immunoblotting, and ELISA as well as with whole cells of AK43 in immunofluorescence. Thus, recombinant 7-4 antibodies expressed in E . coli are structurally and functionally correct.Pseudomonas aeruginosa still remains an important opportunistic pathogen that can cause life-threatening infections in compromised patients including those with severe-burns, cancer, and cystic fibrosis (CF). I Infections are usually localized; however in the acute disease, septicemia occurs and the outcome can be fatal. In fact, P. aeruginosa infections are reported to have had the highest mortality rate among patients with bacteremia over the last 3 decades.2 More recently, many incidences of bacteremia in children with AIDS were also found to be caused by P. aeruginosa, re-emphasizing the significance of this pathogen. Gram-negative septicemia, such as that seen with P. aeruginosa infections, has relied on preventative management and on prophylactic treatments such as antibiotic therapy and immune serum therapy. 1,2 A few anti-Pseudomonas vaccines have been developed based on killed whole cell preparations or lipopolysaccharide (LPS) extracts. 4,5 However, vaccination produces only serotype-specific immunity against P. aeruginosa. 6,7 Treatment with antibiotics is also of limited use due to the intrinsic resistance of P. aeruginosa to these drugs. Although passive immunotherapy minimizes further infection and reduces the risk of septicemia, several of the characterized antibodies are reported to be serotype-specific.9 Therefore, alternative vaccines and therapeutic treatments for P. aeruginosa infections is warranted.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.