BIOTECHNOLOGY FOR OBTAINING THE RECOMBINANT HEAT SHOCK PROTEIN (HSP-60) OF CHLAMYDIA TRACHOMATIS AND EVALUATION OF THE PERSPECTIVES OF ITS USE IN SEROLOGICAL DIAGNOSTICS
Journal Title: Naukovyi visnyk Chernivetskoho universytetu. Biolohiia (Biolohichni systemy) - Year 2014, Vol 6, Issue 2
Abstract
Identification of humoral immune responses to 60 kDa heat shock protein of Chlamydia trachomatis (HSP-60) is of great diagnostic value, as evidence of probable autoimmune process and risk of pathology of female reproductive system. To develop a highly ELISA kit for the detection of anti-HSP-60 antibodies should be used recombinant antigen of the pathogen. A methodological development of biotechnology of recombinant HSP-60 (rHSP-60) with its accumulation in the cytoplasm of E. coli in soluble form has been performed: the optimal parameters of cultivation producer (growth medium, temperature, concentration of expression inducer and biosynthesis duration) providing a preferential (79%) accumulation of the target protein in a soluble form. Purification technology was based on the usage of affinity chromatography on glutathione-sepharose and enzymatic hydrolysis of GST-containing proteins using a clotting factor Xa, and gel filtration on Sephadex G-75 for final purification. We carried out comparative studies of the activity of the obtained rHSP-60 as a part of immunosorbent in ELISA for detecting specific IgG antibodies in human serums. The experiment results for the group of positive serums were assessed based on the positivity index (a ratio of the average arithmetic value of optical density in ELISA for positive and negative (cut off) serums). At the same recombinant protein concentration in the immunosorbent for our obtained rHSP-60, we obtained a 30% higher PI compared to the analogue commercial protein. Of interest were the results of a comparison of the immunological activity of our obtained HSP-60 and its conjugate with GST – positivity indices using these two proteins in the immunosorbent composition were comparable – 2.2 and 2.4, respectively. At the same time, no interaction between serum immunoglobulins and GST enzyme was detected. Such data indicate on absolute possibility of the use of the GST-conjugated rHSP-60 protein for immunodiagnostic purposes. It has been proven that the obtained rHSP-60 preparations as well as its GST-conjugate were highly effective when used in the immunosorbent composition in ELISA for the detection of specific IgG-antibodies.
Authors and Affiliations
O. YU. GALKIN, O. B. BESARAB, A. S. GRYSHYNA, O. M. DUGAN, YU. M. GURZHENKO
Keywords
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