Carcinogenesis is related to the external environment factors and genetic factors. Environmental effect weighs over 50% in cancer risk. The chemicals that cause cancer are defined as chemical carcinogen, and the majority of chemical carcinogens can cause DNA damage. The accumulation of DNA damage can cause gene mutations and increase cancer risk. Thus DNA damage plays an important role in carcinogenesis. Using biomarkers in response to DNA-damage for detection of the existence and exposure dose of chemical carcinogens in environment is one of the important strategies in preventing from cancer.
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, providing all the precursors essential for DNA synthesis. It is the rate-limiting enzyme of both DNA replication and DNA repair. RNR3 is one of big subunit of RNR in Saccharomyces cerevisiae, which has prominent DNA- damage inducible expression effect, and can be developed to be a new biomarker for chemical carcinogen test due to its obvious response to DNA-damage signals. Antibodies are powerful tools in studies on the relationship between the target protein expression level and the induction signals, as well as the base of developing new test method for protein biomarkers. In order to further discover the function and location of yeast RNR3 in vivo and the expression features that induced by DNA damage, we did some preliminary studies in getting monoclonal recombinant antibody with phage display technology. Because of its high conservative structures which may be easily recognized as an autogenous protein, monoclonal antibody is hardly induced against the homologous structure of RNR3 by animal immune system. Whereas genetic manipulated recombinant antibodies to various antigens including self-antigens can be obtained by phage display technology. And the antibodies to RNR3 can help to develop a detection system of chemical carcinogens based on antibody-antigen reactions for biomarker in response to DNA-damage.
In this study, a homologous peptide in yeast RNR3 (loc145-loc298) named RNR3h has been selected after comparing the alignment of the RNR3 to 19 homologues peptides in RNRs from other eukaryotes. RNR3h was expressed in E.coli. The purified RNR3h was uesd as antigen for panning against an immuned phage display antibody library and an unimmuned phage display library. ScFvs (single chain Fragment-variable region) that recognise the RNR3h were obtained and further analyzed. The result shows that the scFvs recognize the conformation antigen epitope, but not liner antigen epitope of RNR3h. The genes of three scFvs have been sequenced and their antigen binding specialities were analyzed. The results showed that the antibodies which could recognize procaryotic expressed protein was not guaranted to recognize eukaryotic expressed protein. Thus, the next step to improve the study is using eukaryotic expressed RNR3h to pan against the phage display antibody library. In addition, two phage display libraries against the C-terminal peptide with different amino acid sequence of yeast RNR1 and RNR3 have been constructed; some preliminary investigations have done on finding the antibodies to the two proteins.