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建立丙型肝炎病毒包膜区基因的真核表达载体,并通过对其序列分析阐明其作为基因接种的可能性。方法:经常规 RT-PCR法从 HCV RNA阳性病人的血清中扩增出 HCV E1区的基因片段,经 EcoR Ⅰ和Xba Ⅰ双酶切后将其克隆到真核表达载体 PcDNA3中,阳性克隆经 Sma Ⅰ和 Xba Ⅰ双酶切鉴定;用双脱氧终止法测序,同时利用计算机软件对其推定的氨基酸序列进行分析。结果:RT-PCR扩增产物经酶切过夜后与经过同样双酶切的真核表达载体 PcDNA3连接,阳性克隆经 Sma Ⅰ和 Xba Ⅰ酶切后产生了预期大小的 144 bp的片段。测序后其核苷酸序列与已经报道的HCVⅠ、Ⅱ、Ⅲ、Ⅳ型的同源性分别为72.8%、93.25%、61.96%和56.44%;其推定的氨基酸序列的同源性分别为 77. 3%、95. 7%、54 6%和 51. 35%;与已经报道的中国株的同源性为 95. 06%和93. 25%;属 HCV Ⅱ型;经计算机辅助分析表明该片段内含有4个可能的糖基化位点, l个跨膜区,其氨基端为信号肽序列,在跨膜区的两端分别含有2个和1个抗原决定簇,而每一个抗原决定簇内均含有1个糖基化位点,该片段内还含有 2个 T细胞识别位点。结论:本实验克隆的 HCV EI基因属Ⅱ型,为我国流行的基因型,其基因的一级结构具备进行有关 HCV核酸免疫的研究条件,其真核表达载体的构建,为进一步研究 HCV EI基因的功能及 HCV基因免疫的研究创造了条件。
To establish a eukaryotic expression vector of hepatitis C virus envelope gene, and to elucidate its possibility of gene inoculation by sequence analysis. Methods: The gene fragment of HCV E1 region was amplified from the serum of HCV RNA-positive patients by routine RT-PCR and cloned into eukaryotic expression vector pcDNA3 by EcoR Ⅰ and Xba Ⅰ digestion. The positive clone Sma Ⅰ and Xba Ⅰ double enzyme digestion; sequencing by dideoxy termination method, at the same time using computer software to deduce the deduced amino acid sequence analysis. Results: RT-PCR products were digested overnight and ligated with the eukaryotic expression vector pcDNA3. The positive clones were digested with Sma Ⅰ and Xba Ⅰ to generate a 144 bp fragment of the expected size. The homologies of the nucleotide sequences of the sequenced strains with those of the previously reported HCV genotypes Ⅰ, Ⅱ, Ⅲ and Ⅳ were 72.8%, 93.25%, 61.96% and 56.44%, respectively. The deduced amino acid sequence homologies were 77. 3%, 95.7%, 546% and 51.35%, respectively. The homology with the reported Chinese strains was 95.06% and 93.25%, which belonged to HCV Ⅱ type. The computer-aided analysis showed that the fragment Contains four possible glycosylation sites, a transmembrane region, the amino-terminal signal peptide sequence, at both ends of the transmembrane region contains two and one antigenic determinant, and each antigenic determinant Contains a glycosylation site within the fragment also contains two T-cell recognition sites. Conclusion: The HCV EI genotype cloned in this study belongs to genotype Ⅱ, which is a popular genotype in China. The primary structure of the gene has the research conditions for HCV nucleic acid immunization and the construction of its eukaryotic expression vector. To further study the HCV EI gene The function and the HCV gene immune research has created the condition.