Construction Of CDNA Library Of Peripheral Blood Lymphocyte Karakul Sheep From Xinjiang And Screening TNF-? Related Genes

In order to study the structure,expression and function of TNF-a of lymphocytes of peripheral blood of Karakul Sheep,this study successfully extracted the Karakul Sheep peripheral blood lymphocytes total RNA and identificated the integrity of the RNA,constructed cDNA library of peripheral blood lymphocyte of Karakul Sheep by Smart cDNA construction kit.and analysed the screening results.First of all,take peripheral blood health sheep,separate white cell,extaction of total RNA,identification quality and determination concentration of the total RNA,synthesis the first chain and the second-d chain DNA by reverse transcription,Using trypsin and SfiI to digest the cDNA and separat and purificate by CHROMA SPIN-400 column.Connect the cDNA with the X TriplEx2 Vector-carrier.Measured the initial concentration,and the amplified library.The results show that:the original storage capacity of of cDNA Library of normal Karakul Sheep is 1.67×106pfu/ml,the amplified library titer is 3.3×109 pfu/ml,the experimental results show that the construction of the cDNA library capacity is accord with library building standards.Second,labeled TNF-a probe and detected the probe labeling efficiency by digoxin labeling kit.The total mass of the TNF-a probe in 20?L is 234ng,the labeled probe was added to 20mL standard hybridization solution at a concentration of 11.7ng/mL,and met the requirements of the subsequent experiments.Last using TNF-? labeled probe to screened the cDNA library of Karakul sheep by nucleic acid hybridization.Successfully screened five TNF-arelated genes from cDNA library including the microtubule transport of molecules interacting gene family,thymosin family gene,a translation factor,ribosome-L37ae gene,elongation factor genes,and do a preliminary analysis,and made the foundation for the further study of the role of genes in the immune response.