Expression and purification of polioviral proteins in E. coli, and production of antisera as reagents for immunological assays

Abstract

Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in Escherichia coli using the autoinduction system, purified, and the proteins were used to raise antisera in rabbits. All antisera detected all three serotypes of PV from infected cell lysates in enzyme-linked immunosorbent assay, immunofluorescence and western blotting.

A novel point mutation (L70P) inactivates poliovirus 3C protease

“…The membrane was washed three times with PBST, and once with PBS, and the expressed proteins were detected using the Pierce ECL reagent (Thermo Fisher Scientific, Bengaluru, India). To confirm the expression of PV proteins in eukaryotic cells, rabbit anti-3AB (1:2,000 dilution) or anti-1AB (1:5,000 dilution) serum (Uma et al, 2016b) was used as primary antibody along with HRP-conjugated anti-rabbit IgG antibody (1:5,000 dilution).…”

“…For gene expression, the HEK293 cells were infected with rAd-3ABCwt or rAd-3ABCmut viruses at a multiplicity of infection (MOI; TCID 50 per cell) of 5. The cells were harvested 24h post-infection, and lysed in Tris-saline (50 mmol/l Tris-HCl [pH 7.5], 100 mmol/l NaCl) containing 1% Nonidet P-40 (NP-40) and 1 mol/l phenylmethyl sulphonyl fluoride (PMSF), and analysed by western blotting using anti-PV3AB polyclonal antibodies (Uma et al, 2016b).…”

“…The loss of protease activity of 3C was further evaluated in a eukaryotic system. Recombinant adenoviruses expressing wild-type (rAd-3ABCwt), L70P mutant (rAd-3ABCmut) 3ABC genes, or no insert (rAd-null) were used to infect HEK293 cells, and protease activity was analyzed by western blot using anti-3AB antibodies (Uma et al, 2016b). Infection of rAd-3ABCwt virus resulted in cleavage of 3ABC, whereas infection with rAd-3ABCmut resulted in expression of uncleaved 3ABC (Fig.…”

“…Polio is on the verge of being eradicated from the globe, and this has resulted in restrictions on handling PV. We have been working on generating reagents in the form of antigens and antibodies (Uma et al, 2016b), which can be used following polio eradication. During this process, we attempted to express the PV 3ABC protein in prokaryotic systems.…”

Expression and purification of human IL-2 protein from Escherichia coli

“…(ii) The production of protein that could be used in serodiagnosis of infections e.g. poliovirus (Uma et al, 2016), and Brucella melitensis (Cloeckaert et al, 2001). (iii) Production of purified proteins that are sold by research companies to be used as positive control in different research experiments e.g.…”