Cloning and expression of HTLV-III DNA

Claim 1

Immunoreactive HTLV-III polypeptide expressed by cells transformed with a recombinant vector containing HTLV-III cDNA.

Claim 25

A method of producing HTLV-III polypeptide, comprising the steps of: (a) cleaving HTLV-III cDNA to produce cDNA fragments; (b) inserting the cDNA fragments into an expression vector to form a recombinant vector; (c) transforming an appropriate host cell with the recombinant vector; and (d) culturing the transformed host cell under conditions sufficient for expression of the polypeptide encoded for by the inserted HTLV-III cDNA.

Claim 26

A method of producing HTLV-III polypeptide, comprising the steps of: (a) cleaving HTLV-III cDNA to produce cDNA fragments; (b) inserting the cDNA fragments into an expression vector to form a recombinant vector; (c) transforming an appropriate host cell with the recombinant vector; and (d) culturing the transformed host cell under conditions sufficient for expression of the polypeptide encoded for by the inserted HTLV-III cDNA.

Claim 28

A method of producing HTLV-III envelope polypeptide, comprising the steps of: (a) cleaving HTLV-III genomic cDNA with the restriction endonuclease Sstl; (b) digesting the cleaved cDNA with restriction endonucleases sufficient to generate restriction fragments which encor.pass at least a portion of the env gene; (c) isolating the restriction fragments; (d) producing DNA fragments of about 200-500 base pairs in length from the restriction fragments; (e) isolating the DNA fragments of about 200-500 base pairs; (f) inserting the isolated fragments into the open reading frame expression vector pMR100 for production of hybrid proteins comprising an env gene product and beta-galactosidase; (g) transforming lac z - E.coli cells with the vector; (h) plating the transformed cells on MacConkey agar plates, maintaining the plates under conditions sufficient for the formation of colonres and selecting cell colonies exhibiting a red color; (i) culturing transformed cells from the selected colonies under conditions which allow expression of the hybrid protein; (j) obtaining cellular protein from the cultured transformed cells; (k) separating the cellular protein obtained; (1) contacting the separated protein with sera frc-n AIDS patients to identify protein which is immunoreactive with the sera; (m) isolating the immunoreactive protein; and (n) optionally separating the env gene expression product from the remainder of the hybrid protein.

Claim 29

A method of producing HTLV-III envelope polypeptide, comprising the steps of: (a) cleaving HTLV-III genomic cDNA with the restriction endonuclease Sstl; (b) digesting the cleaved cDNA with restriction endonucleases sufficient to generate restriction fragments which encor.pass at least a portion of the env gene; (c) isolating the restriction fragments; (d) producing DNA fragments of about 200-500 base pairs in length from the restriction fragments; (e) isolating the DNA fragments of about 200-500 base pairs; (f) inserting the isolated fragments into the open reading frame expression vector pMR100 for production of hybrid proteins comprising an env gene product and beta-galactosidase; (g) transforming lac z - E.coli cells with the vector; (h) plating the transformed cells on MacConkey agar plates, maintaining the plates under conditions sufficient for the formation of colonres and selecting cell colonies exhibiting a red color; (i) culturing transformed cells from the selected colonies under conditions which allow expression of the hybrid protein; (j) obtaining cellular protein from the cultured transformed cells; (k) separating the cellular protein obtained; (1) contacting the separated protein with sera frc-n AIDS patients to identify protein which is immunoreactive with the sera; (m) isolating the immunoreactive protein; and (n) optionally separating the env gene expression product from the remainder of the hybrid protein.

Claim 38

A method of detecting antibodies against HTLV-III in a bodily fluid comprising the steps of: (a) contacting an immunoadsorbent comprising an HTLV-III polypeptide bound to a solid phase with a bodily fluid until antibodies against HTLV-III polypeptide in the bodily fluid bind the solid phase polypeptide; (b) separating the immunoadsorbent from the bodily fluid; (c) contacting the immunoadsorbent with a labeled HTLV-III polypeptide or labeled antibody against human immunoglobulin; and (d) determining the amount of labeled polypeptide bound to immunoadsorbent as an indication of antibody to HTLV-III.

Claim 39

A method of detecting antibodies against HTLV-III in a bodily fluid comprising the steps of: (a) contacting an immunoadsorbent comprising an HTLV-III polypeptide bound to a solid phase with a bodily fluid until antibodies against HTLV-III polypeptide in the bodily fluid bind the solid phase polypeptide; (b) separating the immunoadsorbent from the bodily fluid; (c) contacting the immunoadsorbent with a labeled HTLV-III polypeptide or labeled antibody against human immunoglobulin; and (d) determining the amount of labeled polypeptide bound to immunoadsorbent as an indication of antibody to HTLV-III.

Claim 42

A method of detecting HTLV-III nucleic acid in a bodily fluid (e.g. a cell lysate) comprising the steps of: (a) adsorbing the nucleic acid in a bodily fluid onto an adsorbent; (b) denaturing the adsorbed nucleic acid; (c) contacting the adsorbed nucleic acid with a HTLV-III DNA or RNA probe; and (d) determining if the probe hybridizes with the adsorbed nucleic acid.

Claim 43

A method of detecting HTLV-III nucleic acid in a bodily fluid (e.g. a cell lysate) comprising the steps of: (a) adsorbing the nucleic acid in a bodily fluid onto an adsorbent; (b) denaturing the adsorbed nucleic acid; (c) contacting the adsorbed nucleic acid with a HTLV-III DNA or RNA probe; and (d) determining if the probe hybridizes with the adsorbed nucleic acid.