应用特异性噬菌体抗体克隆和筛选新的大肠癌抗原基因

Cloning and screening novel gene with phage fusion antibody

Objective The cDNA expression library,which was constructed with human colorectal cancer cell HRT-18,was screened with the phage antibody CH209 in order to find novel antigen genes of colorectal carcinoma.Methods Total RNA was extracted from cell HRT-18 and mRNA was isolated from total RNA and then double strand cDNA was synthesized by SMART technique.cDNA was ligated into λ TripIEX2 vector and then was packaged into λ phage in vitro.The primary library was titrated and the percentage of recombinant clones were determined.The length of cDNA inserts was tested for ligation efficiency.The library was screened with phage fusion antibody CH209 and the sequences of the reacted clones were determined.Results The primary library consisted of 6.5×10~(6)pfu/mL,and the percentage of recombinant clones was 97%.The length of cDNA inserts was 0.5-2.0kb.The titer of the amplified cDNA library was 1.1×10~9 pfu/mL.Ten positive clones were obtained and derived from ten different genes.Five of these genes were high homologous to genes known in GenBank,and there were also three genes with low homology to genes known in GenBank.The remainder two genes might be novel genes by matching in GenBank with BLAST software.Conclusion The quality of the constructed cDNA library from human CRC cell HRT-18 is excellent.Ten positive clones were obtained and two of them may be novel genes.