Construction And Screening Of T7 Phage Display CDNA Librariesfrom Liver And Lung Of Microtus Fortis

Schistosoma japonicum is agent of zoonotic infection which can result in serious disease both humans being and in the domestic animal reservoir hosts. Schistosomiasis remains a major public health problem in China. Microtus fortis is the only one mammalian animals with resistance to Schistosomiasis found in the epidemic area in China. Here, we reported the Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis. The liver library was screened to find the schistosomiasis-resistence-related gene of Microtus fortis.1 Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis.1.1 The mRNA was isolated from total RNA from livers and lung of Microtus fortis by Trizol reagent, and used to synthesize the ds cDNA by reverse transcription. Then the ds cDNA was given EcoRâ… and Hindâ…¢adhering ends by ligation with the directional EcoRâ… / Hindâ…¢linkers and digestion with EcoRâ… and Hindâ…¢. The ds cDNA fragments longer than 300bp in length were fractionated by Mini Column, and ligated into the T7 Select 10-3b vertor with EcoRâ… and Hindâ…¢adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library.1.2 The liver library constructed here contained 1.3×10⁷ clones and the titer of the amplied library was 1.8×10¹¹ pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91.7% clones were recombinant and 95.5 % of recombinant clones contained cDNA fragments longer than 300bp in length. The lung library constructed here contained 1.5×10⁶ clones and the titer of the amplied library was 1.1×10¹² pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90 % of recombinant clones contained cDNA fragments longer than 300bp in length. 2 The screening of T7 phage display cDNA library from liver of Microtus fortis with schistosomulua.2.1 The T7 phage display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomulua. Positive clones were identified firstly by PCR and further by sequencing and data analysis through internet BLASTX software of NCBI and Expert Protein Analysis System of expasy and interproscan.2.2 19 available positive clones were obtained and their PCR product sizes ranged from 200～800bp. 13 ESTs, which contain 5 enzymatic activity proteins,5 binding proteins,2 signal transducers and 1 structural constituent protein, were found in NCBI, while 6 ESTs were not found in GenBank. When co-cultured with schistosomulua, the mortality of schistosomulua induced by all of the available positive clones were 1.54-2.60 times than blank control, and 1.06-1.79 times than negative phage control.The results suggested that the T7 phage display cDNA libraries from liver and lung of Microtus fortis were successfully constructed. To Screen The T7 phage display cDNA library from liver of Microtus fortis with schistosomulua,Some of gained clones maight be the schistosomiasis-resistence-related gene of Microtus fortis.