HBx基因的克隆和序列分析及其真核表达载体的构建

CLONING AND SEQUENCE ANALYSIS OF HBX GENE AND ITS EUCARYOCYTIC EXPRESSION VECTOR CONSTRUCTION

目的:通过HBx基因的克隆、序列对比和进化树分析以及pcDNA 3.1(+)-HBx真核表达载体的构建,为进一步研究HBx基因与肝癌发生发展的关系打下基础。方法:用O liga6.0结合P rim er P rem ier 5.0软件设计特异性引物,通过PCR方法从广西HB sA g阳性病人血清中(血清型A drq+)分离得到的病毒株构建的HBxA g质粒中扩增HBx基因。用DNA star5.01和V ector NT I Su ite 8.0软件进行序列对比分析和进化树构建。经双酶切将HBx基因定向插入到pcDNA 3.1(+)质粒中。结果:成功克隆了基因型C型突变型HBx基因,并构建出真核表达载体pcDNA 3.1(+)-HBx。新基因被G eneB ank接受,在G eneB ank上登录号为AY 839630。序列对比和进化树分析表明,新克隆的基因与野生基因型C型有高度的同源性(97.8%),2.2%的变异。结论:从广西HB sA g阳性病人病毒株(血清型A drq+)中发现了新的基因型C型突变型HBx基因(m u tan t genotype C)。pcDNA 3.1(+)-HBx真核表达载体的构建,将为进一步研究HBx基因在树实验性肝癌诱发过程中所起的作用打下基础。

Objective：To further investigate the relations between HBx gene and liver cancer by HBx gene cloning and the construction of pcDNA3.1 （＋）-HBx eucaryocytic expression vector. Methods ： HBx gene was cloned by PCR from HBxAg plasmid constructed from serum of hepatitis B virus positive patients （serotype adrq^＋） of Guangxi. HBx gene was directively inserted into pcDNA3.1 （＋） plasmid by double enzyme cut. HBx gene sequence analysis and its phylogenetic tree were constructed by DNA starS. 01 and vector NTI Suite 8.0 software. Result：HBx gene and pcDNA3.1 （＋）-HBx eucaryocytic expression vector were successfully cloned and constructed. It is a mutant type of wild genotype C of HBx gene. The new gene was accepted and logged in GeneBank, accession number is AY839630. There are 97.8% homogeneity and 2.2% divergency compared with HBx gene wild type C by alignment and phylogenetic tree analysis. Conclusion： we had cloned HBx gene mutant genotype C from serum of HBV positive patients. The construction of pcDNA3. 1 （＋）-HBx eucaryocytic expression vector will lay down the foundation for further investigate the role of HBx gene in development of the experimental liver cancer on treeshrew.