Expression, purification, and human antibody response to a 67 kDa vaccine candidate for schistosomiasis japonica.
Schistosomiasis remains a leading cause of morbidity and mortality in the developing tropical world, and vaccines to prevent these infections remain a scientific and public health priority. Sj67 is a 67 kDa Schistosoma japonicum surface membrane protein homologous to a family of actin-binding proteins. Sj67 is recognized by a mouse monoclonal antibody (mAb 6) that confers resistance to challenge infection in passive transfer experiments. These data support Sj67 as a potential vaccine candidate for schistosomiasis japonica. In the present study, we report the ligation-independent cloning of a cDNA encoding thioredoxin/elastin-like polypeptide (ELP)/rSj67 into a pET-32 Xa/LIC vector. Soluble recombinant fusion protein (Thio-ELP-rSj67) was expressed and purified using anion-exchange and size exclusion chromatography. rSj67 was cleaved from the Thio-ELP fusion partner by digestion with Factor Xa protease and purified using hydroxyapatite column chromatography. Endotoxin was reduced by absorption to a polymyxin support. Purified rSj67 had a molecular weight of 67 kDa and N-terminal sequencing confirmed that the first five amino acids of the recombinant protein matched the predicted sequence for the Sj67 gene. In Western blot analysis, rSj67 was recognized by the Sj67 specific mAb 6 antibody. IgG antibodies in sera from schistosomiasis infected volunteers living in an endemic area of the Philippines (n = 13) recognized rSj67 with 4.7-fold greater median fluorescence compared to uninfected North American controls (n = 5) (p < 0.009). Together, these data confirm the expression and purification of recombinant Sj67 and its immuno-reactivity with sera from S. japonicum infected humans.
Solomon
Julie S
JS
International Health Institute, Brown University, Providence, RI, USA.
Nixon
Christian P
CP
McGarvey
Stephen T
ST
Acosta
Luz P
LP
Manalo
Daria
D
Kurtis
Jonathan D
JD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000917:Antibody Formation / Q000276:immunology; D000947:Antigens, Helminth / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D015870:Gene Expression; D015801:Helminth Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D012549:Schistosoma japonicum / Q000235:genetics / Q000276:immunology; D012554:Schistosomiasis japonica / Q000097:blood / Q000276:immunology / Q000401:mortality / Q000517:prevention & control; D022223:Vaccines, Subunit / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D015801:Helminth Proteins; D007074:Immunoglobulin G; C103355:JF antigen, Schistosoma japonicum; D008840:Microfilament Proteins; D022223:Vaccines, Subunit