Construction and identification of T7 phage display cDNA library of PK15 cells

【Objective】To research the interaction between swine viruses and their host cell, T7 phage display cDNA library of PK15 cells was developed. 【Method】The mRNA was extracted from PK15 cells and its quality was identified using aldehyde-agarose gel electrophoresis. The double-strand cDNA was synthesized by reverse transcription. EcoR I and Hind III linkers were linked to the double-strand cDNA after the cDNA terminal was flushed. Then the modified cDNA was digested by EcoR I and Hind III enzyme. After that, cDNA was linked into the T7select vector arms. We used the phage packaging extract to package the linked vector in vitro for the construction of the preliminary T7 phage display cDNA library, which was identified by tittering and PCR. 【Result】The titer of library was tested by plaque assay. The titer of primary library and amplified library were 2.0×105PFU/mL and 6.0×1010PFU/mL, respectively. PCR identification results showed that the recombination ratio was about 95.83%,and the inserts varied from 500 bp to 2000 bp(500-750 bp: 21.73%;750-100 bp: 21.73%; 1000-2000 bp: 39.13%). Sequencing results of the 20 clones selected randomly from the library suggested that all of them were homologous with the swine sequence. 【Conclusion】The library had a good quality of capacity and recombination rate for further researches on the interaction between swine viruses and their host cell, T7 phage display cDNA library of PK15 cells.