HBx基因缺失型突变体HBx-d382与HBx-d431原核表达载体的构建和鉴定

Construction of Prokaryotic Expression Vector Carrying HBx-d382 and HBx-d431 Genes from Hepatocellular Carcinoma Tissue

目的为探讨HBx基因缺失型突变体HBx-d382和HBx-d431在临床上应用的价值,构建HBx基因缺失型突变体HBx-d382和HBx-d431原核表达载体。方法以pGM-T/HBx-d382和pGM-T/HBx-d431质粒为模板,PCR扩增含EcoRⅠ和XhoⅠ酶切位点的HBx基因片段。HBx基因PCR扩增产物与载体pET-28a(+)经双酶切、连接,形成重组DNA后转化到大肠杆菌BL21(DE3)中,经卡那霉素筛选、酶切鉴定和DNA序列测定,筛选出重组HBx基因缺失型突变体原核表达载体。结果成功构建了重组pET-28a(+)/HBx-d382和pET-28a(+)HBx-d431原核表达载体,重组前后HBx基因片段序列一致。结论pET-28a(+)/HBx-d382和pET-28a(+)/HBx-d431原核表达载体构建成功,为HBx基因缺失型突变体HBx-d382和HBx-d431蛋白的免疫原性和临床应用的研究奠定了基础。

Objective To construct the prokaryotic expression vector carrying HBx-d382 and HBx-d431 genes for exploring the relationship between HBx deletion mutant and hepatocellular carcinoma. Methods HBx-d382 and HBx-d431 genes with EcoR I and Xho I endoenzyme sites were obtained by using PCR. The genes were cloned into pGM-T and produced pGM-T/HBx-d382 and pGM-T/HBx-d431 plasmids. The plasmids were transferred to E. coli BL21 （DE3）. Recombinant plasmid sequence were subsequently verified by EcoR Ⅰ and Xho Ⅰ endoenzyme digestion and auto-sequencing assay. Results We have constructed the prokaryotic expression vector of HBx-d382 and HBx- d431 carrying the integrated HBx-d382 or HBx-d431 gene fragment. Conclusion pET-28a （＋）/HBx-d382 and pET- 28a（＋）/HBx-d431 prokaryotic expression vectors can be used for the study of HBx-d382 and HBx-d431 proteins.