Title:
PREPARATION OF CDNA LIBRARY AND SCREENING OF POLYPEPTIDE
Document Type and Number:
Japanese Patent JP3229590
Kind Code:
B2
Abstract:
PROBLEM TO BE SOLVED: To obtain an efficiently retrievable cDNA library by preparing a cDNA fragment from an object cell, linking different restriction enzyme sites to both ends of the fragment including a signal peptide followed by PCR and then cleavage and by integrating the resulting fragment.
SOLUTION: This cDNA library is obtained by the following procedure: a single-stranded cDNA corresponding to mRNA isolated from an object cell is synthesized using a random primer, an oligo dT is added to the 3'-end of the single-stranded cDNA thus formed, and the corresponding double-stranded cDNA is synthesized using, as primer, a poly A oligomer to which a specific restriction enzyme (enzyme I) site is linked; next, the double-stranded cDNA is cleaved, and the resulting fragments are fractionated based on their size, and then a linker containing a site of a specific restriction enzyme (enzyme II) differing from the enzyme I is linked to the resulting fragment followed by PCR with a primer containing the enzyme site and then lyzing the amplified product with the enzyme I-II, the resulting fragment is linked to a site upstream of a protein gene deprived of a signal, peptide and integrated into a vector for eukaryocytes to effect transformation.
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Inventors:
Tasuku Honjo
Akira Tashiro
Hideaki Tada
Akira Tashiro
Hideaki Tada
Application Number:
JP29524598A
Publication Date:
November 19, 2001
Filing Date:
October 16, 1998
Export Citation:
Assignee:
Tasuku Honjo
Ono Pharmaceutical Co., Ltd.
Ono Pharmaceutical Co., Ltd.
International Classes:
A61K38/00; A61K31/00; A61K39/395; A61P7/00; A61P7/06; A61P35/00; A61P35/02; C07K14/00; C07K14/47; C07K14/475; C07K14/52; C07K14/53; C07K14/535; C07K14/705; C07K14/715; C07K16/00; C12N1/21; C12N15/09; C12N15/10; C12N15/12; C12N15/62; C12N15/63; C12P21/02; C12P21/08; C12Q1/68; C12R1/19; (IPC1-7): C12N15/09
Other References:
Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，Ｖｏｌ．１９，Ｎｏ．２５，ｐ．７０９７−７１０４（１９９１）
Attorney, Agent or Firm:
Landlord Kunihisa