【摘要】 目的比较不同载体和不同大小的目的基因片段对戊型肝炎病毒(HEV)基因免疫抗原表达的影响,为基于HEV中和抗原表位的HEV基因免疫提供一定的依据。方法将含Ⅳ型HEV中国株中和抗原表位的p166和p179片段编码基因分别克隆入pTR421和pCDNA3.1两种真核表达载体,用脂质体介导基因转染HepG2人肝癌细胞系,经间接免疫荧光和Western blot分析以及将质粒注射小鼠局部肌肉组织后经免疫组织化学染色检测,分析目的基因在体内外的表达水平。结果成功构建了pTR421-166、pTR421-179、pCDNA3.1-166和pCDNA3.1-179四种重组质粒,经限制性内切酶双酶切和核苷酸测序鉴定,编码基因正确无误;pTR421-179转染的细胞以及注射小鼠的局部肌肉组织可以检测到p179的表达,而pCDNA3.1-179、pCDNA3.1-166以及pTR421-166均检测不到目的基因在体内、外的抗原表达。结论载体和目的基因片段的选择显著影响HEV抗原在体内、外的表达,直接关系到基因免疫的成功与否。更多还原

【Abstract】 AIM: To investigate the effects of different vectors and gene fragments on antigen expression of hepatitis E virus (HEV) DNA immunization. METHODS: Gene fragments encoding p166 and p179, which contain the neutralization antigenic epitopes of a Chinese strain of HEV genotype Ⅳ, were cloned into two different eukaryotic expression vectors (pTR421 and pCDNA3.1), respectively. The in vitro expression level of p166 and p179 in HepG2 cells transfected by each of the recombinant plasmids with lipofectamine2000 was examined by means of immunofluorescence and Western blot. Meanwhile, the in vivo expression level in muscles of mice was examined with immunohistochemistry staining. RESULTS: Four recombinant plasmids, pTR421-166、 pTR421-179、 pCDNA3.1-166 and pCDNA3.1-179, were constructed successfully and confirmed correct with restriction endonuclease analysis and nucleotide sequencing. The antigen expression was only detected in HepG2 cells transfected by pTR421-179 and in myocytes of the mice injected with pTR421-179. Neither in vitro nor in vivo antigen expression was detected with pTR421-166 although p166 was only 13 amino acids shorter than p179 at N terminus. Neither pCDNA3.1-166 nor pCDNA3.1-179 was expressed in vitro and in vivo. CONCLUSION: Selection of the vectors and gene fragments is critical to HEV gene expression and HEV DNA vaccine.[更多还原