Superscript choice system for cdna synthesis

Example 3

Identification of Antigen Protein to which MMG49 Antibody Binds

An antigen protein to which the MMG49 antibody bound was identified by an expression cloning method.

First, a cDNA library was generated from MM.1s cells, to which the MMG49 antibody was known to bind, using a superscript choice system for cDNA synthesis (Invitrogen), and was inserted into a pMXs retrovirus vector (donated by Professor Toshio Kitamura at the Institute of Medical Science of the University of Tokyo) using a BstXI adaptor (Invitrogen). The thus generated cDNA library was introduced into plat-E cells (donated by Professor Toshio Kitamura), and BaF3 cells were infected with the resultant retrovirus. Thus, BaF3 cells expressing an MM.1s-derived cDNA library were obtained.

Next, the cells were repeatedly concentrated by staining the cells with the MMG49 antibody and sorting positive cells by FACS (FIG. 3). After the third sorting, most cells were cells that bound to the MMG49 antibody. Then, the retrovirus insert carried by those cells was amplified by PCR, and then sequenced to identify its base sequence. As a result, it was revealed that the insert carried by the cells was ITGB7.

Example 3

Identification of Antigen Protein to which MMG49 Antibody Binds

An antigen protein to which the MMG49 antibody bound was identified by an expression cloning method.

First, a cDNA library was generated from MM.1s cells, to which the MMG49 antibody was known to bind, using a superscript choice system for cDNA synthesis (Invitrogen), and was inserted into a pMXs retrovirus vector (donated by Professor Toshio Kitamura at the Institute of Medical Science of the University of Tokyo) using a BstXI adaptor (Invitrogen). The thus generated cDNA library was introduced into plat-E cells (donated by Professor Toshio Kitamura), and BaF3 cells were infected with the resultant retrovirus. Thus, BaF3 cells expressing an MM.1s-derived cDNA library were obtained.

Next, the cells were repeatedly concentrated by staining the cells with the MMG49 antibody and sorting positive cells by FACS (FIG. 3). After the third sorting, most cells were cells that bound to the MMG49 antibody. Then, the retrovirus insert carried by those cells was amplified by PCR, and then sequenced to identify its base sequence. As a result, it was revealed that the insert carried by the cells was ITGB7.