猪Utx慢病毒过表达/干扰载体的构建和病毒包装

Construction and Package of Porcine Utx Lentviral Overexpression/interference Vectors

UTX(ubiquitously transcribed TPR gene on the X chromosome)作为H3K27me2/3的去甲基酶,能够调控动物发育过程,并有着重要的生物学作用。根据猪Utx预测序列,扩增Utx的CDS区全长序列。利用Utx的CDS区序列信息,构建pCD513B-CMV-Utx过表达载体,设计并合成干扰shRNA和阴性对照寡核苷酸片段。其中干扰shRNA及阴性对照通过退火形成双链DNA,并插入pCD513B-U6慢病毒干扰载体。采用双酶切的方法和DNA测序鉴定重组载体。通过干扰和过表达载体质粒共转293T细胞,利用qRT-PCR筛选出最有效的干扰序列。重组质粒与其他3种慢病毒辅助包装质粒(pRsv-Rev、pGag-Pol、pVSV-G)共转染293T细胞包装慢病毒。结果表明,本试验成功扩增Utx基因的全长序列,且成功构建了Utx的过表达和干扰载体。qRT-PCR筛选出pCD513B-U6-Utx-shRNA-3干扰效率最显著,干扰效率为70%。本试验成功包装出慢病毒Lenti-Utx-shRNA、Lenti-NC和Lenti-Utx,测定的病毒滴度分别为6.1×107 TU/mL、4.8×107 TU/mL和3.9×105 TU/mL。此试验为进一步在猪源细胞中进行Utx基因的研究奠定基础。

UTX（ubiquitously transcribed TPR gene on the X chromosome）acts as H3K27me2/3demethylase and plays important roles in regulating animal developmental processes.According to predicted Sus scrofa Utx gene sequence in the NCBI,complete CDS sequence of Utx was amplified.Subsequently,the pCD513B-CMV-Utx overexpression vector was constructed,and 5interference shRNA and 1negative control oligonucleotide fragments was designed by using the acquired Utxcomplete CDS sequence.The interference shRNAs and negative control oligonucleotide were annealed to form double strand DNA,which were inserted into pCD513B-U6 lentiviral interference vector.The recombination vectors were confirmed by double-enzyme digestion and DNA sequencing.Then interference and overexpression vectors were cotransfected into 293 Tcell line.The most efficient interference sequence was screened out according to qRT-PCR results.In addition,the 293 Tcells were transfected by recombined plasmids and three kinds of package plasmids（pRsv-Rev,pGag-Pol,pVSV-G）to package lentivirus.Results showed that the complete Utx gene sequence were successfully amplified.Utx overexpression and interference vectors were successfully constructed.Meanwhile,qRT-PCR results suggested pCD513B-U6-Utx-shRNA-3 was most efficient and the interference efficiency reached 70%.Finally,Lenti-Utx-shRNA,Lenti-NC and Lenti-Utx were successfully packaged,which titers were 6.1×107 TU/mL,4.8×107 TU/mL and 3.9×105 TU/mL respectively.Taken together,these results make foundation for the funetionel studies of the Utx gene in porcine cells.