| In this paper,we used SMART technology to build a multi-wave sheep cDNA library of peripheral blood lymphocytes,lebeled IL-8 probe,proceeded nucleic acid hybridization screening,and analyzed the results.First,using Trizol method to extract Duolang sheep lymphocyte total RNA,by reverse transcription to synthesis the first strand cDNA,double-stranded cDNA obtained after LD PCR amplification,then by proteinase K and Sfi I digestion,joints and small fragments generated by CHROMA SPIN-400 column chromatography,centrifugation,collected more than 400 bp cDNA fragments,and then ligated these exogenous fragments with λTriplEx2 Vector and conducted its package,determined the nitial titer of the library.The results showed that the initial titer of the cDNA library constructed was5.6×107pfu/mL,the clone library titer was 1.5×1010pfu/mL.cDNA library constructed was in line with the capacity of future research requirements.We built a duolangsheep peripheral blood lymphocyte cDNA library can be easily screened desired target gene,more important,it can be used to isolate full-length genes and do gene function studies.Second,positive clones were sequenced,received seven IL-8 immune-related genes.Analysed the nucleic acid sequence by DNAtools,and NCBI BLAST to perform nucleic acid and protein blast.the results indicated the genes have the domain of glycosyl phosphatidy linositol(GPI),coenzyme Q-cytochrome C reductase(UCR)ubiquitin,EF-h calcium-binding motifs(EF-H),heme protein(Globin),ribosome(Ribosomal),and all these genes play its role in the immune response. |
