乙型肝炎病毒X蛋白激活基因1的克隆化与序列分析

Cloning and identifications of human gene 1 transactivated by hepatitis B virus X antigen

目的:利用分子生物学技术,研究乙型肝炎病毒(HBV)X蛋白(HBxAg)的反式激活作用,克隆HBxAg反式激活作用的靶基因,为进一步探索HBxAg的反式激活作用,以及反式激活作用的靶基因,阐明HBV感染引起慢性肝炎、肝细胞癌(HCC)发生发展的分子生物学机制,为探索新型预防和治疗技术、寻求新型途径奠定理论基础。方法:利用聚合酶链反应(PCR)技术,扩增HBxAg的编码基因,构建表达载体pcDNA3.1(-)-X,转染肝母细胞瘤细胞系HepG2,与转染空白载体的HepG2细胞对照组分别提取总mRNA,并进行抑制性消减杂交(SSH)分析。对于获得的差异表达基因片段序列的同源性基因进行搜索,确认为与已知的功能基因无同源性之后,利用表达序列标签(EST)序列的搜索和比对,进行电子拼接,完成新基因序列的确定。然后自HepG2细胞提取总mRNA,应用生物信息学技术确定的新基因的序列设计特异性引物,进行逆转录多聚酶链反应(RT-PCR)技术的扩增,获得阳性克隆之后,进行鉴定并对克隆的基因及其编码产物的序列进行分析。结果:PCR技术扩增获得的HBxAg基因序列,经过限制性酶切鉴定和序列测定证实无误。转染HepG2细胞并提取足量的mRNA,利用SSH技术进行分析,获得的基因片段序列分析结果表明,其中之一为新型基因片段序列,与GenBank中注册的已知功能基因序列没有同源性。通过对EST数据库中注册的基因片段序列同源性的搜索和比对,电子拼接成功,根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列,确定新型基因序列。从HepG2细胞提取总mRNA,以RT-PCR技术,扩增获得该新基因的全基因序列,并测序证实,命名为XTP1,在GenBank中注册,注册号为AF488828。结论:利用分子生物学技术与生物信息学技术相结合,发现并鉴定、克隆了HBxAg的反式激活作用的新基因XTP1,并为进一步研究HBxAg的反式激活作用的分子生物学机制和探索新型治疗技术奠定了基础。

AIM: To study the transactivation effects of HBxAg,and clone the target genes of HBxAg transactivating effects,in order to help understand the mechanism of pathogenesis of HBxAg. METHODS: Polymerase chain reaction(PCR)was employed to amplify the coding sequence of HBxAg.The hepatoblastoma cell HepG2 was transfected by pcDNA3.1(-)and pcDNA3.1 (-)-X,respectively.Total mRNA was purified from the HepG2 cells transfected and suppression subtractive hybridization (SSH)method was used to analyze the differentially expressed DNA sequence between the two groups.The sequences were searched for homologous DNA sequence from GenBank. The new DNA sequence was confirmed and the full-length coding sequence was identified according to the Kozak rule and the existence of polyadenyl signal sequences.Reverse transcription PCR(RT-PCR)was used to amplity the new gene by using mRNA from HepG2 cell as the template.The coding sequence for the new gene was deduced according to the nucleotide sequence. RESULTS: PCR technique was employed to amplify the coding sequence for HBxAg by using pCP10 plasmid containing whole HBV genome as the template. The recombinant plasmid expressing HBxAg was confirmed by restriction enzyme digestion and sequencing. HepG2 cells were trans fected with pcDNA3.1(-) and pcDNA3.1(-)-X by lipofectamine, respectively. Total mRNA was purified from transfected HepG2 cell, and suppression subtractive hybridization method was used for the screening and identification of differentially expressed genes by these two cell groups. After sequencing, each DNA sequence was compared with the genes deposited in the GenBank and the new gene with no homology with known genes in this database was identified. Electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene and in conjunction with Kozak rule and the existence of polyadenyl signal sequence. RT-PCR technique was used to amplify the new gene, named as XTP1, from the mRNA of HepG2 cells. The sequence for the XTP1 gene was deposited into GenBank, and the accession number is AF488828. CONCLUSION: A new gene named XTP1 which is transac- tivated by hepatitis B virus X protein has been successfully cloned by molecular biological methods. These results pave the way for the study of the molecular mechanism of HBxAg transactivating effects and the development of new therapy for chronic hepatitis B.