公开(公告)号：JPH0833485A

公开(公告)日：1996-02-06

申请号：JP32954091

申请日：1991-11-18

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  OHASHI SHINICHI

IPC: C12N11/00 ,  C12N9/06

Abstract: PURPOSE:To obtain a highly active immobilized enzyme for bioreactor, etc., in high efficiency by introducing an amino acid sequence having a cysteine residue at the terminal to the carboxy-terminal of an enzyme protein and immobilizing the modified protein to a carrier through an SH group. CONSTITUTION:This immobilized enzyme having high uniformity and activity and useful as an element of a bioreactor, etc., is produced by constructing an expression vector coding a modified enzyme protein obtained by introducing a short amino acid sequence to the carboxy terminal of an enzyme protein (e.g. dihydrofolate reductase) to form cysteine residue on the carboxy terminal of the protein, introducing the expression vector into a host cell, culturing the transformed cell to effect the expression of the gene and obtain a modified enzyme protein having an amino acid sequence composed of several number of amino acid residues having a cysteine residue terminal on the carboxy terminal of the enzyme protein and bonding the modified protein to an immobilization carrier through a mercapto group of the cysteine residue at the carboxy terminal.

公开(公告)号：JPH05339295A

公开(公告)日：1993-12-21

申请号：JP34867791

申请日：1991-12-05

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  OHASHI SHINICHI

IPC: C07K16/00 ,  C07K14/00 ,  C07K19/00 ,  C12N15/09 ,  C12N15/62 ,  C12P21/02 ,  C12R1/19 ,  C07K15/14

Abstract: PURPOSE:To produce an antibody by bonding the objective peptide to the carboxyl terminal of dihydrofolic acid reductase(DHFR) and using the produced fused protein as an immunizing antibody. CONSTITUTION:The objective process for the preparation of an antibody against a peptide is composed of a process for producing a DHFR fused protein having the objective peptide on the carboxyl terminal side by genetic engineering technique, a process for separating the objective fused protein from the host microorganism and purifying the protein and a process for immunizing an animal using the objective highly purified fused protein.

公开(公告)号：JPH05308992A

公开(公告)日：1993-11-22

申请号：JP10592092

申请日：1992-03-31

Applicant: AGENCY IND SCIENCE TECHN

Inventor： KOKUBU TOMOKUNI ,  IWAKURA MASAHIRO ,  OHASHI SHINICHI

IPC: A61K39/395 ,  C12N5/10 ,  C12N5/20 ,  C12N15/02 ,  C12N15/06 ,  C12P21/08 ,  C12R1/91 ,  G01N33/53 ,  G01N33/577

Abstract: PURPOSE:To obtain monoclonal antibodies against leucine enkephalin. CONSTITUTION:The monoclonal antibodies RIPT36 and RIPT74 against leucine enkephalin(LEK) and a hybridoma cell capable of producing the antibodies. Uniform anti-LEK monoclonal antibodies can always be obtained in a required sufficient amount and the resultant antibodies can be used to extremely specifically analyze the LEK present in a living body specimen, etc., according to various methods and applied to the diagnostic medical field, etc.

公开(公告)号：JPH05236990A

公开(公告)日：1993-09-17

申请号：JP35083591

申请日：1991-12-11

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  OHASHI SHINICHI

IPC: C12N5/10 ,  C07K16/00 ,  C07K16/44 ,  C12N5/20 ,  C12N15/02 ,  C12N15/06 ,  C12N15/09 ,  C12N15/62 ,  C12P21/02 ,  C12P21/08 ,  C12R1/19 ,  C12R1/91 ,  G01N33/531 ,  G01N33/577

Abstract: PURPOSE:To simply form the monoclonal antibody against a compound having a low antigen-activity. CONSTITUTION:In the formation of a specific monoclonal antibody, a complex dihydrofolic acid reductase (DHFR) having the objective compound on the side of its carbonyl terminal is formed, and an animal to be immunized is immunized, sensitized with the reductase (DHFR), and further subjected to the screening of the monoclonal antibody with the complex DHFR to produce a hybridoma cell producing the monoclonal antibody against the objective compound. The cell obtained as the result of the screening is used to form the objective monoclonal antibody.

公开(公告)号：JPH04117284A

公开(公告)日：1992-04-17

申请号：JP12320190

申请日：1990-05-15

Applicant: AGENCY IND SCIENCE TECHN ,  HITACHI CHEMICAL CO LTD

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TANAKA YOSHIO ,  BABA KENZO ,  IZUTSU HIROSHI ,  OBARA KAZUHIKO ,  TAKASUKA AKIKO

IPC: C12N15/09 ,  A61K38/00 ,  A61K38/44 ,  A61P37/08 ,  C07K1/22 ,  C07K14/00 ,  C07K19/00 ,  C12N1/21 ,  C12N15/12 ,  C12N15/53 ,  C12N15/62 ,  C12P21/02 ,  C12R1/19

Abstract: NEW MATERIAL:A recombinant plasmid containing a base sequence encoding dihydrofolate reductase-antiallergic peptide fused protein. EXAMPLE:Plasmid pBBK10MM. USE:Production of DSDGK. PREPARATION:DNAs shown by formula II and formula III are chemically synthesized and a duplex DNA shown by formula TV is obtained by annealing. Recombinant plasmid pMEK2 is cleft with BamHI and XhoI to give a DNA fragment, which is linked to the duplex DNA shown by formula TV with T4-DNA ligase to give new plasmid pBBK10MM.

公开(公告)号：JPH02142479A

公开(公告)日：1990-05-31

申请号：JP29691388

申请日：1988-11-24

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C12N15/09 ,  C12N1/21 ,  C12N15/16 ,  C12N15/62 ,  C12N15/63 ,  C12P21/02 ,  C12R1/19

Abstract: NEW MATERIAL:A recombinant plasmid pPRLh4, stably replicative in Escherichia coli, capable of imparting trimethoprim and ampicillin tolerance to host Escherichia coli and having a DNA sequence expressed by the formula. USE:For mass-producing prolactin(PRL). PREPARATION:For example, a gene phpRL78 capable of coding PRL is cleaved with a restriction enzyme HindIII and the cleave part is smoothed with a DNA polymerase. The resultant gene is subsequently cleaved with a restriction enzyme BstEII to prepare a DNA fragment, which is then linked to a plasmid pPRL1-13 treated with a restriction enzyme XhoI using a T4-DNA ligase. The obtained conjugate is then inserted into Escherichia coli and the resultant transformant is cultured to select a train capable of producing DHFR-PRL and afford the objective recombinant plasmid pPRLh4.

公开(公告)号：JPH02142476A

公开(公告)日：1990-05-31

申请号：JP29420488

申请日：1988-11-21

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  OHASHI SHINICHI ,  TANAKA YOSHIO

IPC: C12N15/09 ,  C07K14/00 ,  C07K14/575 ,  C07K14/60 ,  C07K19/00 ,  C12N1/21 ,  C12N15/18 ,  C12N15/62 ,  C12P21/02 ,  C12R1/19

Abstract: NEW MATERIAL:A recombinant plasmid pGRFM44-6 stably replicated in Esherichia coli, capable of giving trimethoprim resistance and amplicillin resistance to the Escherichia coli as a host and coding the fused protein of a bovine growth hormone-releasing factor derivative having an amino acid sequence of the formula with a dihydrofolic acid reductase. USE:For the production of a bovine growth hormone-releasing factor (GRS) and a dihydrofolic acid reductase(DHFR). PREPARATION:For example, a recombinant plasmid pSG1-12 coding DHFR and the sequence of Np.1-29 amino acids of bovine GRF is treated with a restriction enzyme. The produced DNA fragment is combined with a synthetic DNA coding the sequence of No.29-44 amino acids of the GRF and subsequently inserted into an Escherichia coli, followed by culturing the Esherichia coli and screening the cultured colony thereof the select a clone producing DHFR-GRF, thereby providing the pGRFM44-6.

公开(公告)号：JPS6387981A

公开(公告)日：1988-04-19

申请号：JP23400386

申请日：1986-09-30

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  KOKUBU TOMOKUNI ,  FURUSAWA KIYOTAKA ,  TSUDA KEISHIRO

IPC: C12N15/09 ,  C07K14/665 ,  C12N1/20 ,  C12N1/21 ,  C12N9/06 ,  C12N15/00 ,  C12P21/02 ,  C12R1/19

Abstract: PURPOSE:To provide a recombinant plasmid containing integrated gene of dihydrofolic acid reductase (DHFR)-leucine encephaline fused protein, stably replicable in E.coli and capable of imparting said host with trimethoprim resistance and ampicillin resistance. CONSTITUTION:Plasmid pBSDHFR1 is cut with restriction enzymes EcoRI and PstI and separated by 1% agarose gel electrophoresis to obtain a small fragment DNA. Separately, plasmid pBR322 is cut with restriction enzyme BamHI and Pst and separated by 1% agarose gel electrophoresis to obtain a large fragment DNA. The DNA of formula I and formula II are synthesized, the 5'-terminal is converted to phosphoric acid group and the product is annealed to obtain a DNA coding leucine encephaline. The above 3 kinds of DNA molecules are linked together, introduced into E.coli C600 strain and cultured. The objective recombinant DNA containing the sequence of formula III coding a DHFR-leucine encephaline fused protein can be separated from the obtained colony.

公开(公告)号：JPS6328394A

公开(公告)日：1988-02-06

申请号：JP17045986

申请日：1986-07-19

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO ,  TSUDA KEISHIRO

IPC: C12N15/09 ,  C12N1/20 ,  C12N1/21 ,  C12N15/00 ,  C12N15/70 ,  C12P21/00 ,  C12R1/125 ,  C12R1/19

Abstract: PURPOSE:The titled plasmid, obtained by replacing a dihydrofolate reductase gene part of plasmid pTP64-1 with a dihydrofolate reductase gene of Bacillus subtilis, stably replicable in a host and capable of imparting trimethoprim and ampicillin resistance. CONSTITUTION:A plasmid pTP64-1 is cleaved with a restriction enzyme to replace a dihydrofolate reductase part with dihydrofolate reductase of Bacillus subtilis to give the aimed plasmid pBSDHFR1. The resultant novel recombinant plasmid can be stably replicated in Escherichia coli (E. coli) to impart trimethoprim and ampicillin resistance to the E. coli which is a host. The plasmid has a size of 5504 base pairs and DNA sequence shown in the figure (No.501 and thereafter are omitted) and is useful in the fields of microbial, pharmaceutical industries.

公开(公告)号：JP2000247999A

公开(公告)日：2000-09-12

申请号：JP4955999

申请日：1999-02-26

Applicant: AGENCY IND SCIENCE TECHN

Inventor： IWAKURA MASAHIRO

IPC: C07K17/00 ,  C12N11/00

Abstract: PROBLEM TO BE SOLVED: To improve immobilizing yield of a protein in a method for immobilizing the protein through the carboxyl group of carboxy terminal of the protein to a carrier by utilizing amino bond forming reaction through a cyanocysteine residue. SOLUTION: In immobilization of a protein represented by formula I: NH2-R1- COOH (R1 represents arbitrary amino acid residue chain), a protein represented by formula II: NH2-R1-CO-NH-CH(CH2-SCN)-CO-NH-R2-COOH (R1 has the above meaning; R2 represents chain of arbitrary amino acid residue rich in acidic amino acid, strongly negatively charging in nearly neutral and bringing isoelectric point of the compound of formula II to acidity) is reacted with an immobilizing carrier represented by formula III: NH2-Y (Y represents an immobilized carrier having a primary amine as a functional group) to produce the objective immobilized protein represented by formula IV: NH2-R1-CO-NH-Y (R1 and Y have the above meanings).