Purification of heat labile toxin from Bordetella pertussis vaccine strain 134 employed indigenous technology

Abstract

Aim and objective: The aim of the study was the evaluation of purified HLT from B. pertussis vaccine strain 134 by employing indigenous technology and examining the immuno-biochemical aspects of the purified protein.
Materials and methods: Shaker cultivation of B. pertussis strain 134, sterility, opacity confirmation, TCA precipitation of cellular proteins, G50 purification  subsequent DEAE purification, purity analysis, specific activity of HLT, and immune response analysis.
Results: The shaker cultivated B. pertussis strain 134 passed its quality attributes such as sterility, opacity and purity. During TCA precipitation the B. pertussis desired proteins were precipitated and confirmed. The indigenous bed height was optimized and recovery was also calculated in G50 purification. The fractions were analyzed for OD, the total protein concentration and the results were 0.074–0.214, and the total protein content was found between 12.33 lg/ml and 35.67 lg/ml. Subsequent DEAE purification of selected G50 fractions was done and the fractions 9 and 14 had higher OD values of 0.675 and 0.397. Furthermore the DEAE purified samples were structurally analyzed through SDS PAGE and it was found that HLT in the single polypeptide band was around 140 kDa. The qualitative immune response analysis of DEAE purified selected fractions pool showed positive immune response in ODD assay, and in the case of guinea pig antisera it led to the development of diagnostic kits such as ELISA and other vital techniques. Here, in case of guinea pig experiment, the hemorrhagic necrosis analysis showed the necrosis on the skin at the injection site.
Conclusion: The B. pertussis HLT could be purified through two phase with G50 and DEAE, cost effective techniques, the G50 purification has reduced the bioburden problems during DEAE purification and at the same time the quality of the product was high.

Keywords: Heat labile toxin; Pertussis; Whole cell pertussis vaccine; DEAE; Whooping cough