[Construction and identification of the cDNA phage expression library for human colorectal cancer antigens].

OBJECTIVE

To construct a cDNA phage expression library for human colorectal carcinoma antigens.

METHODS

After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA.

RESULTS

The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp.

CONCLUSION

The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.