HBx基因的克隆和序列分析及其真核表达载体的构建

目的：通过HBx基因的克隆、序列对比和进化树分析以及peDNA3．1（＋）-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Oliga6．0结合Primer Premier5．0软件设计特异性引物，通过PCR方法从广西HBsAg阳性病人血清中（血清型Adrq^＋）分离得到的病毒株构建的HBxAg质粒中扩增HBx基因。用DNA star5．01和Vector NTI Suite8．0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pcDNA3．1（＋）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pcDNA3．1（＋）HBx。新基因被GeneBank接受，在GeneBank上登录号为AY839630。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（97．80A），2．2％的变异。结论：从广西HBsAg阳性病人病毒株（血清型Adrq^＋）中发现了新的基因型C型突变型HBx基因（mutantgenotypeC）。peDNA3．1（＋）-HBx真核表达载体的构建，将为进一步研究HBx基因在树鼩实验性肝癌诱发过程中所起的作用打下基础。

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

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 star5.01 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.