Construction of T7 phage display library from Cryptosporidium parvum

To screen protective antigen gene of Cryptosporidium parvum,the T7 phage display library from C.parvum was constructed,and the total RNA was extracted from C.parvum by Trizol reagent.The mRNA was isolated from total RNA by PolyATract mRNA Isolation Kit and the ds cDNA was synthesized by reverse transcription.In addition the directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoRⅠand HindⅢ,which resulted in ds cDNA with EcoRⅠand HindⅢ ends.And the ds cDNA fragments longer than 400bp in length were fractionated by Mini Column,and then ligated into the T7 Select 10-3b vertor with EcoRⅠand HindⅢ ends.After packaging in vitro,the T7 Select 10-3b vertor was transformed into BLT5403 to construct the T7 phage display library.The experimental results showed that the library contained 1.2×10~7 clones,and approximately 96.7% of the library were recombinant.The titer of the amplied library was 2.4×10~(10) pfu/mL.The cDNA fragments longer than 400bp in length were 92% of 100 plaques by PCR identification.These results suggests that the T7 phage display library from C.parvum is constructed successfully.