Recombinant, biologically active human zone pellucida proteins 3 (hzp3) to test male fertility
AssigneeEastern Virginia Medical School
InventorsKE-WEN DONG, SERGIO OEHNINGER, WILLIAM E. GIBBONS
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Abstract
The invention relates to methods for producing recombinant human zona pellucida protein (“rhZP3”) and glycosylated peptide having biological activity of binding to human spermatozoa. A human ovarian cell line is used to produce rhZP3 having a glycosylation pattern required for full biological activity. Methods of determining useful peptides with binding activity for human sperm and their syntheses, as well as using such peptides and proteins in therapeutics and diagnostics are discussed.
Claims21
A purified recombinant glycopolypeptide of about 65 kd to about 100 kd that comprises approximately 40% to 60% carbohydrate by weight and that can strongly bind human spermatozoa and induce an acrosome reaction in the spermatozoa.
A process for producing a glycopolypeptide having the biological activity of hZP3 protein, comprising the steps of:
- (a) transducing a cell from a human ovarian cell line with a polynucleotide that encodes polypeptide comprising SEQ ID NO:
- 1;
- (b) establishing a stable-transfected cell culture for producing the glycopolypeptide;
- and
- (c) isolating the glycopolypeptide from the cell culture.
A glycopolypeptide having between 41 and 400 amino acids and having an active portion that preferentially binds to human sperm, wherein the active portion comprises an amino acid sequence that is more than 54% homologous with SEQ ID NO:
- 1 and has a predicted O-glycosylation site at a serine five positions from the carboxyl terminus of the active portion.
A method of detecting male infertility from a human spermatozoa sample, comprising the steps of:
- (a) contacting purified recombinant glycopolypeptide of about 65 kd to about 100 kd that comprises approximately 50% carbohydrate and that can strongly bind to human spermatozoa and induce an acrosome reaction, with spermatozoa from the sample to form an admixture;
- (b) detecting biological activity of the spermatozoa in the admixture;
- and
- (c) comparing the biological activity with a reference value.
A nucleic acid vector useful for producing a glycopolypeptide smaller than 300 amino acids long, wherein the glycopolypeptide specifically binds to human sperm and the vector codes for an active amino acid sequence portion that is more than 54% identical with SEQ ID NO:
- 1.
A human ovarian cell containing a nucleic acid vector useful for producing a glycopolypeptide less than 300 amino acids long, wherein the glycopolypeptide strongly binds to human sperm and the vector codes for an amino acid sequence that has a portion which is more than 75% identical with SEQ ID NO:
- 1.
Dependencies are inferred from the claim text.