摘要
目的:构建HBV核心蛋白与人类载脂蛋白B mRNA编辑酶催化多肽3C(A3C)融合蛋白HBV载体质粒。方法:构建表达HBV核心蛋白的乙肝病毒载体质粒PdssX;以质粒PC-A3C为模板扩增A3C,将扩增的A3C及PdssX分别用Aor13H Ⅰ和BSE Ⅱ酶切双酶切,以T4连接酶连接转化TOP10感受态细胞,然后PCR和双酶切鉴定及测序鉴定。结果:酶切所获载体片段和目的基因片段的大小均与预期的相一致,经测序证实为pCH-CoreA3c基因,表明CoreA3c基因HBV载体质粒pCH-CoreA3c构建成功。结论:成功构建HBV核心蛋白与A3C融合蛋白HBV载体质粒pCH-CoreA3c,为下一步CoreA3c融合蛋白的原核表达及其抗病毒作用研究奠定了基础。
Objective To construct a recombinant plasmid expressing HBV core antigen and apolipoprotain B mRNA-editing enzyme catalytic polypeptide-like3C（A3C） fusion protein. Methods HBV core antigen expression plasmid PdssX was first constructed. A3C gene was amplified from plasmid PC-A3C by polymerase chain reaction （PCR）. A3C and PdssX were digested by endonucleases Aor13H I and BSE II , respectively, then the digested products were connected by T4 Ligase and transformed into TOP10 chemical competent cells. The reeombinants were identified by PCR, double restriction endonuclease digestions and DNA sequencing. Results The result of agarose gel electrophoresis showed that the sizes of digested vector fragment and cloned gene fragments were consistent with expectations, and the result of DNA sequencinge showed that the fusion gene sequence was correct. Conclusion The plasmid pCH-CoreA3C expressing HBV core antigen and A3C fusion protein was constructed successfully, which lay a good condition for further research on CoreA3C expression and inhibiting the replication of hepatitis B virus.
出处
《实用医学杂志》
CAS
北大核心
2009年第24期4106-4109,共4页
The Journal of Practical Medicine
