[Feasibility to use of recombinant domains of immunoglobulin-like protein LigA as a promising marker for serological testing of patients with leptospirosis].
OBJECTIVE
To clone the DNA fragment encoding conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein.


METHODS
E. coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes--according to "Qiagen company's protocols. Extraction and purification of proteins were performed using original method.


RESULTS
DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E. coli. Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit.


CONCLUSIONS
Recombinant domain of LigA in chimeric protein produced in E. coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.
Sharapova
N E
NE
Galushkina
Z M
ZM
Poletaeva
N N
NN
Lavrova
N V
NV
Verkhovskaia
L V
LV
Lunin
V G
VG
Petrov
E M
EM
Samsonova
A P
AP
Anan'ina
Iu V
IuV
D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006801:Humans; D007921:Leptospira interrogans / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; C487005:LigA-m protein, Leptospira interrogans; D011994:Recombinant Proteins