Screening And Identification Of The Gene Of Human Lung Cancer-associated Antigen

Objective: When exogenous genes were linked with reasonable expression vectors, the recombinants could express the relevant objected proteins. Applied the specific antibody which could response with the proteins could screen the interested genes or respective segments from cDNA library. All these will be benefit with immunologic diagnosis and immunotherapy for neoplasm. The mAb N-35 derived from lymphocyte hybridoma technique could specific combine with diverse carcinoma cells but not with normal cells. So it is very important to isolate the antigen gene which corresponding to mAb N-35. Method: Immunoscreening for the detection of reactive clones in the H128cDNA expression library, which was constructed in expression vector lambda gt11 with SCLC cell line H128 by using mAb N-35. Then subcloning was performed after obtaining the positive clones. And additional work was made to identify these clones by PCR and Western blot. Result: 42 positive clones encoding antigen genes were obtained after immunoscreening. One was selected and subcloned from these clones, sequenced it was 1262bp,and know it is not full length of cDNA molecule. Sequence data of the cDNA molecule indicates that there was 99% homology with human "proteinphosphatase 2, catalytic submit alpha isoform" gene in Genbank. Western blot experiment showed that the expression product of the cDNA segment keeps specific response with mAb N-35. Conclusion: Obtained a cDNA segment of human lung cancer associated antigen, it has highly homologous with the human "protein phosphatase 2 , catalytic subunit alpha isoform" gene, and mutation of this gene and the protein coded by it maybe tumorigenesis . Additional we found the method of defined the real positive clone by Western blot.