|Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
The aim of study was to isolate AA4.1 cDNA.To do so, we first tried to purify AA4.1 molecules using anti-AA4.1 antibody conjugated affinity chromatography. The yield rate of the affinity-purified AA4.1 was too small for us to determine an amino acid sequence of AA4.1 fragment by the method. In order to determine an optimal condition to solubilize AA4.1 molelcules, cell surface proteins of a murine AA4.1-positive pre-B cell line IIB4.2.1, were first biotinylated, and then immunoprecipitated with anti-AA4.1. However, no antibody-specific band was detected on SDS-PAGE.These results indicate that AA4.1 molecules were too labile to keep a stable antigenisity when they were solubilized in detergent of as much as 1% digitonin solution.
Next, we performed an expression cloning of AA4.1 cDNA.This method was used because of the following reasons, 1) AA4.1 molecules expressed on a cell surface were their native form through expression cDNA cloning ; 2) Therefore, we thought AA4.1 would be quite po
ssibly recognized by the anti-AA4.1 antibody. cDNA were synthesized from 2 mug mRNA of IIB4.2.1, and recombinated into pCDM8 expression plasmid vector. 1.4x10^6 clones of directional recombinant plasmid library (A) were constructed with 1.0kb to 2.0 kb-sized cDNA.4x10^5 clones of derectional cDNA library (B) were synthesized from cDNAs of more than 2.0 kb. Each library was divided into about 20 groups. Plasmids which were purified from each group were transfected into COS 7 cells by DEAE-dextran method for the first time and by protoplast fusion procedure for the second time, and possible AA4.1 positive transfectants were collected by MACS and FACS.The pCDM8 plasmids which were obtained by screening more than 3 times by the procedure described above, were examined to see if each plasmid clone had AA4.1 cDNA in it. We screened 8.5x10^4 clones from the library (A) and 1.9x10^5 clones from the library (B). However, We have so far obtained no positive clone.
In conclusion, we have not yet succeeded in cloning of AA4.1 cDNA.There are a few reasons for this unpredicted results. First, AA4.1 epitopers that ant-AA4.1 antibody recognized were so labile. Second, the lability made it difficult for us to purify AA4.1 molecules. We are still investigatinf for a better purification method for AA4.1 molecule and for expression cDNA cloning procedure. Less