Cloning Of SNRPB Gene From Sika Deer And Construction Of Eukaryotic Expression Vector

SNRPB(Small Nuclear Ribonucleoprotein Polypeptides B)is the core element of the spliceosome Small Nuclear Ribonucleo protein,can coordinate the spliceosome splicing.Some recent studies have revealed that SNRPB can affect a large number of biological processes related to cancer,such as RNA processing,DNA repair,and so on,which are closely related to the growth of cancer cells.Cancer is one of the most serious diseases threatening human health,cancer cells can proliferate infinitely and rapidly,but the proliferation speed of antler growth center cells in the rapid growth period is more than 30 times than that.if the mechanism of antler proliferation is understanded,and apply it to cancer treatment,it may provide new ideas and directions for cancer treatment.In this study,the homologous primers of SNRPB gene were designed according to thegene sequence of cattle in Genbank.We adopted RT-PCR technology and DNA molecular cloning technology to obtain the cDNA sequence of sika deer SNRPB gene successfully.The relative expression level of SNRPB gene among early,mid and late growth periods from mesenchymal tissue were determined using Real time PCR technology.At the same time,PEGFP-C1-SNRPB was constructed and transfected into 293T cells.Using real-time fluorescence quantitative PCR to study over expression level of SNRPB gene.The experimental results are as follows:1.In the study the length of SNRPB gene sequence was 783 bp,which encodes 240 amino acids and has a typical LSM domain.By comparing nucleotide sequences,it was found that the sika deer SNRPB gene has a high homology with Odocoileus virginianus,Bos taurus and Ovis aries(99%,97%and 97%,respectively),which indicated that the gene is relatively conserved in evolution.The phylogenetic tree was constructed and showed that the evolution distance between sika deer and Odocoileus virginianus,Ovis aries and Bos taurus was close,that between sika deer and Sus scrofa,Monodon monoceros was slightly far,and between sika deer and pan paniscus,camelus ferus and Marmota flaviventris was far,which also conforms to the classical taxonomic view.2.The relative expression levels of SNRPB gene in the mesenchymal tissues of deer antler were detected by real-time fluorescence quantitative PCR.The result showed that the expression level was the highest in the early stage and the lowest in the middle stage earlier.3.After construction of eukaryotic expression vector and recombinant eukaryotic expression vector pEGFP-C1-SNRPB and empty vector were transiently transfected into 293T cells,a large number of green fluorescence was observed under fluorescence microscope.Real time PCR showed that the relative expression of SNRPB gene in experimental group was about 367 times of that in the control group.This indicated that the eukaryotic expression vector of SNRPB gene was successfully expressed in 293T cells.