US 2003/0165523 A1 · Truncated recombinant major outer membrane protein antigen (R56) of orientia tsutsugamushi strains Karp, Kato and gilliam and its use in antibody based detection assays and vaccines

Abstract

A recombinant, refolded non-fusion polypeptide expressed from a truncated r56 gene of the causative agent of scrub typhus, Orientia tsutsugamushi for the Karp, Kato and Gilliam strains has been produced. The invention is useful for detecting prior exposure to scrub typhus, screening for and/or identification of at least one infectious strain-similarity (i.e. a Karp-like, Kato-like or Gilliam-like strain) based on its strength of reaction toward a truncated protein and as a component in vaccine formulations and production of immune globulins for passive prophylaxis and immunity in subjects.

Claims33

Dependent claims

A recombinant polypeptide which comprises the amino acid sequence SEQ ID NO.:

A process for producing a recombinant polypeptide which comprises the following steps:

a) transforming a plasmid carrying the r56 insert into the expression host E. coli BL21;
b) inducing recombinant E. coli expressing r56 with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the log phase and propagated in LB medium overnight at 37° C. with shaking;
c) expressing the r56 polypeptides as inclusion bodies (IB) in E. coli BL21;
d) re-suspending the cell pellets in a 20 mM Tris-HCl buffer (Buffer A) having a pH of 8.0 and contains 5 mM ethylene diamine tetraacetic acid (EDTA) and 0.1 mM of phenylmethylsulfonyl fluoride (PMSF);
e) disrupting the cells by passing them through a microfluidizer 3 times;
f) centrifuging the cell extract at 8,000× g for 30 minutes
g) extracting the pellets with 2 Molar urea in the said buffer of step d.;
h) dissolving the pellets produced in step g in 8 M urea containing 10 to 20 mM DTT for about 20 minutes;
i) centrifuging the product of step h at 8,000× g for minutes;
j) applying the clear supernatant formed in step i to size-exclusion columns TSK P3000 SW (21.5 mm×50 cm)-tandem TSK P4000 SW (21.5 mm×100 cm)column equilibrated with 8 M urea and 1 mM DTT in 20 mM Tris-HCl, pH 7.8 (buffer B)
k) pooling and loading peak fractions containing the r56 polypeptide onto the non-exchange DEAE column (21.5 mm×30 cm)
l) eluting the bound r56 formed in steps with a linear gradient of Nacl from zero to 0.4 M in the said buffer B for about 30 to 60 minutes at a flow rate of 5 ml/minute;
m) refolding the r56 in the said buffer B by three sequential dialysis with 6 M urea, 4 m urea and 2 M of urea, respectively in said buffer A alone;
n) combining and dialyzing the peak fractions of the r56 against 8 volumes of 6 M urea in said buffer A for 30 minutes at 4° C. followed by 2 changes of the dialysis solution and dialyzing the product for 60 minutes;
o) dialyzing the product of step n with 4 M urea, containing 0.3 micro M of the oxidized form of glutathione followed by dialyzing with 2 M urea in buffer A;
p) dialyzing the product of step o against said buffer A, with 2 initial changes of buffer, for 30 minutes;
q) dialyzing the product of step p overnight at 4° C. and
r) separating the product of step q.

15 more elements

Dependencies are inferred from the claim text.

Truncated recombinant major outer membrane protein antigen (R56) of orientia tsutsugamushi strains Karp, Kato and gilliam and its use in antibody based detection assays and vaccines

Life of this patent

Truncated recombinant major outer membrane protein antigen (R56) of orientia tsutsugamushi strains Karp, Kato and gilliam and its use in antibody based detection assays and vaccines

Life of this patent

Abstract

Claims33