Studies On The Construction Of CDNA Expression Library From Adult Fasciola Gigantica And Its Expressed Sequence Tags

Fasciola gigantica always parasitizes the ruminants and mainly distributes the east and south of china,and always be harmful to the yield-power of stockbreeding and the health of people,but so far there are no efficacious drugs and vaccines to control its developmemt,so, in order to find out the latent vaccine molecules to resist it,the cDNA expression library from adult Fasciola gigantica was constructed,and the EST mothed was used to analyse the obtainted sequences.A high-quality Fasciola gigantica cDNA library was constructed by using the SMART technique, the result showed that the unamplified library capacity was 1.08 X106 Pfu/ml, the library recombination rate was 96.6%,the titer of the amplied library was 2.41 X 10~9Pfu/ml and the average inserted cDNA fragment was about 1000bp. Then 40 clones were selected to sequence from the BM25.8 plasmids,32 sequences obtainted from the plasmids were proved to be useful after editing using some biosoftwares.After analysising by using the BLASTx and BLASTn programs,9ESTs were proved to be the known-genes, and 16 ESTs whose aligment value were very low or which suited sequence were not found were proved to be unknown-genes.The results also showed that the 9 ESTs represent the cystein protease-, eggshell protein and calcium-binding protein,the other 16 unknown ESTs included some signal conductive genes protein translative genes and immune stimulative genes and so on. One EST was choosed to in silico cloning by using the bioinformatic and some biosoftwares on internet,one contig whose length was 1920bp% ORF was 930 bp and coded 310 amino acidwas obtained ,and was predicted to be eggshell protein gene.In order to prove the dependability of the in silico cloning method ,one primer which intend to amplify to 758bp was designed along the contig, the experiment cloned the target sequence successfully and the sequencing result was identical to the target sequence absolutely.It also beared out the cDNA library can provide the resources for cloning new genes.The study showed that the Fasdola gigantica cDNA library was constructed firstly and the strategy of how to clone the gene by in silico cloning mothed based the internet databases efficiently was explored,and had important implications for further studies on controling Fasdola gigantica diseases.