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US 2003/0166057 A1Published application
Method and apparatus for the production of soluble MHC antigens and uses thereof
OwnerWilliam H. Hildebrand, Kiley R. Prillimaninventors
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Abstract
The field of the invention relates in general to at least one method and apparatus for the production of soluble MHC antigens and more particularly, but not by way of limitation, to at least one method and apparatus for the production of soluble Class I and II HLA molecules. The field of the invention also includes such produced soluble Class I and II HLA molecules and their use. According to the methodology of the present invention, the soluble Class I and II HLA molecules can be produced from either gDNA or cDNA starting material.
Claims44
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- isolating HLA allele mRNA from a source and reverse transcribing the mRNA to obtain allelic cDNA;
- amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer having a stop codon incorporated into a 3′ primer thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules.
3 more elements
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- isolating HLA allele mRNA from a source and reverse transcribing the mRNA to obtain allelic cDNA;
- amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic CDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules.
3 more elements
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- isolating HLA allele mRNA from a source and reverse transcribing the mRNA to obtain allelic cDNA;
- amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules.
3 more elements
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- obtaining gDNA which encodes a HLA allele;
- amplifying the allelic gDNA by PCR, wherein the amplification utilizes at least one locus-specific primer having a stop codon incorporated into a 3′ primer thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic gDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding the soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules.
3 more elements
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- obtaining gDNA encoding a HLA allele;
- isolating HLA allele mRNA from gDNA and reverse transcribing the mRNA to obtain allelic cDNA;
- amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules.
4 more elements
A method for the production of soluble HLA molecules in a cell pharm having an appropriate growth media therein, comprising the steps of:
- obtaining gDNA encoding a HLA allele;
- isolating HLA allele mRNA from gDNA and reverse transcribing the mRNA to obtain allelic cDNA;
- amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
- inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
- electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
- and
- inoculating the cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules.
4 more elements
A multimeric HLA complex, comprising:
- a substrate;
- and
- at least two soluble HLA molecules attached to the substrate.
Dependencies are inferred from the claim text.