[Preparation and property study of the recombinant Camel anti-SpaA-N single domain antibodies].
OBJECTIVE
In order to obtain single domain antibody against surface protective antigen A (SpaA)of Erysipelothrix rhusiopathiae.


METHODS
The SpaA-N recombinant protein was used to screen binders from Bactrian camel VHH phage display library. After sequencing,  the interested VHH gene fragments were subcloned into pET-30a vector to overexpress the protein in E.coli BL21. The binding specificity of the recombinant VHH with SpaA-N was determined by Western blotting. The thermal stability of single-domain antibody was evaluated by ELISA.


RESULTS
By enrichment of screening,  2 clones were selected.  Recombinant single domain antibodies purified by Ni-ion affinity chromatography showed a single band at M(r); 29 000, 23 000 on SDS-PAGE. ELISA results showed that VHH can bind its antigen specifically. After thermal denaturation,  VHH can restore the antigen binding ability after refolding. Western blotting results showed that the recombinant VHH specific bind surface protective antigen of Erysipelothrix rhusiopathiae at M(r);  66 000. Two VHH single domain antibodies with high thermal stability and good antigen binding specificity were identified by screening Bactrian camel VHH phage display library.


CONCLUSIONS
Two single domain antibodies that specifically aggulated SpaA-N is obtained,  which provide the basis for further study in the immune role of single domain antibody against Erysipelothrix rhusiopathiae infection.
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. wxx605@sohu.com
Qing-xia
QX
Lu
Ge-le-tu
GL
Qing
Chun-jing
CJ
Yang
Xue-qin
XQ
Xia
Jiang-wei
JW
Li
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002162:Camelus / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C114457:SpaA protein, Erysipelothrix rhusiopathiae