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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 investigate the clinical value of HBx-d382 and HBx-d431 in HBx-deficient mutants, we constructed HBx-d382 and HBx-d431 prokaryotic expression vectors. Methods HBx gene fragment containing EcoR Ⅰ and Xho Ⅰ restriction sites was amplified by PCR using pGM-T / HBx-d382 and pGM-T / HBx-d431 as templates. HBx gene was amplified by PCR and cloned into vector pET-28a (+). The recombinant plasmid was transformed into E.coli BL21 (DE3). After screening by kanamycin, enzyme digestion and DNA sequencing, The prokaryotic expression vector of recombinant HBx gene deletion mutant was screened out. Results The recombinant prokaryotic expression vector pET-28a (+) / HBx-d382 and pET-28a (+) HBx-d431 were successfully constructed. The sequence of the HBx gene before and after recombination was consistent. Conclusion The prokaryotic expression vectors pET-28a (+) / HBx-d382 and pET-28a (+) / HBx-d431 were successfully constructed and were used for immunogenicity and clinical application of HBx-d382 and HBx-d431 proteins. The research laid the foundation.