Production in Escherichia coli, purification and immunogenicity of acrosomal protein SP-10, a candidate contraceptive vaccine.
The testis-specific human sperm antigen, SP-10, has been designated a 'primary vaccine candidate' by the World Health Organization Taskforce on Contraceptive Vaccines. Molecular cloning and sequencing of the cDNAs coding for human (h) and baboon (b) SP-10 have been reported. To produce large amounts of pure antigen for ongoing studies of the immunogenicity and anti-fertility effects of SP-10, we used an efficient Escherichia coli expression system. The full-length open reading frames for hSP-10 and bSP-10 were placed under the inducible T7 bacteriophage RNA polymerase/promoter system. An in-frame fusion was made such that a His6 stretch was produced at the C terminus of SP-10. Upon induction of gene expression, large amounts of hSP-10 or bSP-10 were synthesized and the recombinant (re-) protein segregated into an insoluble fraction. The protein was then solubilized in 6 M guanidine.HCl and purified by immobilized metal affinity chromatography (IMAC). The yield of purified bSP-10 preparation was approx. 20 micrograms/ml of culture. Immunoreactivity of the purified re-SP-10 with MHS-10, a monoclonal antibody specific to SP-10, and rabbit polyclonal sera raised against SP-10, indicated that the synthesized antigen was suitable for immunization studies. Four female baboons were then immunized with the re-bSP-10 antigen. Immunoblots using pre-immune and immune sera from these animals indicated that all four baboons produced antibodies that reacted with native SP-10 extracted from human sperm in a manner identical to that of MHS-10, the positive control. Immune sera also stained the acrosome region of human and baboon sperm heads by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)
Reddi
P P
PP
Dept. of Anatomy and Cell Biology, University of Virginia, Charlottesville 22908.
Castillo
J R
JR
Klotz
K
K
Flickinger
C J
CJ
Herr
J C
JC
D000177:Acrosome; D000818:Animals; D000941:Antigens / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003269:Contraception, Immunologic; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005260:Female; D012739:Gonadal Steroid Hormones / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010215:Papio; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
C512729:ACRV1 protein, human; D000941:Antigens; D018076:DNA, Complementary; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D014614:Vaccines, Synthetic