摘要
目的 构建乙型肝炎病毒（HBV）X基因高效真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及乙型肝炎病毒母婴垂直传播的可能机制奠定基础.方法 设计并合成乙型肝炎病毒X基因的引物,以我国急性肝炎患者血清中获得的全长乙型肝炎病毒序列C基因型作为构建载体X基因序列的母板进行扩增,将扩增产物X基因连接到真核表达载体pcDNA3.1（＋）上,酶切图谱分析、PCR检测和扩增产物序列分析等鉴定所构建的真核表达载体.结果 以重组载体为模板、用乙型肝炎病毒X基因引物PCR扩增得到的目的 片段为500bp,与已知乙型肝炎病毒X基因大小相同,酶切后发现在500bp左右及5kb左右出现两条特异性条带,分别与已知X基因序列相同.结论 成功构建了乙型肝炎病毒X基因真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及宫内感染的可能机制奠定了基础.
Objective To construct a highly effective eukaryotic expression vector pcDNA3.1 （ ＋ ） with HBV X gene[ pcDNA3.1 （ ＋ ） -X1 , and then to study the function of HBV X gene and the probable mechanism of intrauterine infection. Methods Primers of HBV X gene were designed. All HBV sequences genotype C as mother blank, HBV X gene was amplified by PCR. The PCR products were connected to the eukaryotic expression vectors pCDNA3.1 （ ＋ ） by restriction endonucleases and T4 DNA joining enzyme. Restriction endonucleases digest analysis, PCR analysis, and sequence analysis were used to identify the recombinants. Results Taking reconstructed vector as mould,the fragment amplified by PCR was 500bp,which was same as known HBV X gene. After treated with restriction endonucleases, two specific bands presented around 500bp and 5kb, which were same as know X gene sequence. Conclusion The eukaryotic expression vector pCDNA3. 1 （ ＋ ）-X has been successfully constructed, which provides some foundation for analysis of the function of HBV X gene and probable mechanism of intrauterine infection.
出处
《中国妇幼健康研究》
2011年第3期301-303,共3页
Chinese Journal of Woman and Child Health Research
基金
国家自然科学基金资助项目（NO.30872220）
关键词
乙型肝炎病毒
X基因
真核表达载体
宫内感染
hepatitis B virus （HBV）
X gene
eukaryotie expression vector
intrauterine infection
