淋巴细胞内与乙型肝炎病毒X蛋白结合的新蛋白X-30编码基因的克隆

Screening and cloning of a novel gene coding for X-30 protein interacting with hepatitis B virus X antigen in lymphocyte

目的:为探讨 HBxAg 在 HBV 致病过程中的作用,筛选并克隆人淋巴细胞 cDNA 文库中与 HBxAg 有相互作用的蛋白基因.方法:利用酵母双杂交系统3筛选并克隆人淋巴细胞 cDNA文库中与 HBxAg 有相互作用的蛋白的基因.将 HBxAg 编码基因连接入酵母表达载体 pGBKT7中构建诱饵质粒,转化酵母细胞 AH109并在其内表达,然后与转化了人淋巴cDNA 文库质粒 pACT2的酵母细胞 Y187进行配合,双重筛选阳性菌落,提取质粒并测序,结果进行生物信息学分析,发现其中有1个未知基因.根据 GenBank 中的序列信息设计引物,从 HepG2细胞提取总 RNA,以逆转录多聚酶链反应(RT-PCR)技术扩增获得该新基因的全长序列,并测序证实,命名为 X-30,在 GenBank 中注册,注册号为AY280722.结果:X-30基因的编码序列全长为315个核苷酸(nt),编码产物由103个氨基酸残基(aa)组成.结论:HBxAg 与淋巴细胞结合蛋白新型基因 X-30的筛选与克隆,为进一步研究 HBxAg 的分子生物学机制及探索新型治疗技术奠定了基础.

AIM:To investigate biological functions of hepatitis B virus X antigen(HBxAg)protein by screening and cloning the target genes in lymphocytes interacting with HBxAg. METHODS:The yeast-two hybrid technique was performed to seek proteins in lymphocytes interacting with HBxAg. HBxAg bait plasmid was constructed by ligating the HBxAg gene with carrier plasmid pGBKTT,and then transformed into yeast AH109(a type).The transformed yeast cells was amplified and mated with yeast cells Y187(α type) containing lymphocytes cDNA library plasmid pCAT2 in 2x YPDA medium.Diploid yeast cells were plated on synthetic dropout nutrient medium and selected two times.Plasmid of true positive blue colonies were extracted and analyzed by DNA sequencing and blast in GenBank.The integrity sequence of a new gene X-30 was amplified from the mRNA of HepG2 cell by reverse transcription PCR.The sequence for the HBx-30 gene was deposited into GenBank, and the accession number is AY280722. RESULTS:The full-length coding sequence of X-30 was consisted of 315 nucleic acid and 103 amino acid residues. CONCLUSION:These results will pave the way for the study of the molecular mechanism of the transactivating effects of X protein of HBV and the development of new therapy for chronic hepatitis B.