HBV X区基因部分序列的亚克隆与PCR检测

Subclone of partial sequence of gene of HBV X region and PCR detection

目的:探讨HBV X区基因用于乙型病毒性肝炎早期诊断价值和意义。方法:设计特异性引物,将pBR322-HBV质粒X区部分序列PCR产物AT亚克隆至pBS-T载体,提取和纯化质粒DNA,再对HBV X区序列进行PCR扩增。结果:获得了预期希望的质粒。PCR的最佳退火温度为51℃,灵敏度达到101拷贝/2μl,线性范围101-1010拷贝/2μl。讨论:pBR322-HBV质粒中的靶基因成功地被亚克隆至pBS-T载体。pBR322-HBV中X区目的序列扩增产物为57bp,该小片段勿需纯化就可直接AT亚克隆至新载体,有利于后续的常规PCR检测和TaqMan MGB荧光定量PCR检测。

Objective： To explore the significance and value of HBV X region genes in the early diagnosis of Hepatitis B. Methods： The partial gene sequence of HBV X region in pBR322 - HBV was amplified by PCR. The products amplified by PCR were sub - cloned into the pBS -T vector,which were an effective AT clone plasmid, q3ae positive AT sub- clones were sequenced. Results： The expected plasmid with HBV X region gene was obtained, The optimal annealing temperature was 51 ℃, The sensitivity reached 101 copies per 2μl. The linear range was 101 - 1010 copies per 2μl. Disolssion： The target genes in pBR322 - HBV were sub - cloned to pBS - T. The amplification product of the target gene sequence in pBR322 - HBV was 57bp. It can be directly sub-cloned to the new vector without purification, which favors the routine PCR and TaqMan MGB fluorescent quantitative PCR for the early detection of HBV.