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目的 :对轮状病毒 SA1 1株 VP7基因进行修饰 ,使其表达后锚定于宿主细胞膜表面 ,同时构建一个可使目的蛋白分泌或跨膜表达的载体。方法 :用 PCR的方法把 VP7天然信号肽替换为流感病毒血凝素信号肽。然后 ,在基因下游添加流感病毒血凝素跨膜区编码序列 ,从而构建在宿主细胞表面表达的轮状病毒 SA1 1 VP7基因。利用遗传密码简并性在信号肽序列 3′端和跨膜区 5′端分别引入 Sty 和 Eco RV限制性内切酶识别位点 ,从而实现分泌 /跨膜基因修饰载体的构建。把野生型和跨膜型 VP7基因分别克隆入 pc DNA3哺乳动物细胞表达载体 ,转染 Hela细胞 ,得到稳定表达两种 VP7基因的细胞系。对外源基因在细胞染色体上的整合情况以及基因表达情况进行检测。结果 :酶切鉴定和 DNA序列分析表明膜锚定型 VP7基因和分泌 /跨膜基因修饰载体的构建正确。PCR和免疫荧光实验证明 ,VP7基因已经整合进入宿主细胞染色体 ,野生型和膜锚定型 VP7基因表达后分别定位于宿主细胞质和细胞膜。结论 :成功地构建了可在细胞膜表面锚定表达的 VP7基因 ,为进一步研究轮状病毒基因工程疫苗奠定了基础。新型分泌 /跨膜基因修饰载体为研究蛋白合成后加工和提高其免疫原性提供了有力的工具。
OBJECTIVE: To modify the VP7 gene of SA1 1 strain of rotavirus so that it can be anchored to the surface of host cell membrane after it is expressed, meanwhile, to construct a vector capable of secreting or transmembrane the target protein. Methods: VP7 native signal peptide was replaced by influenza virus hemagglutinin signal peptide by PCR method. Then, the coding sequence of the transmembrane region of influenza virus hemagglutinin was added downstream of the gene to construct the rotavirus SA1 1 VP7 gene expressed on the surface of the host cell. The secretory / transmembrane gene-modified vector was constructed by introducing the restriction sites of Sty and Eco RV into the 3 ’end of the signal peptide sequence and the 5’ end of the transmembrane region, respectively, using the degeneracy of the genetic code. The wild-type and transmembrane VP7 genes were cloned into pcDNA3 mammalian cell expression vector respectively and transfected into Hela cells to obtain a cell line stably expressing two VP7 genes. The integration of exogenous genes in the chromosomes of the cells and the gene expression were tested. Results: Enzyme digestion and DNA sequence analysis showed that the membrane-anchored VP7 gene and secretory / transmembrane gene-modified vector were constructed correctly. PCR and immunofluorescence experiments showed that the VP7 gene has been integrated into the host cell chromosome, and the wild-type and membrane-anchored VP7 genes were localized in the host cytoplasm and the cell membrane, respectively. Conclusion: VP7 gene, which can be anchored on the surface of cell membrane, has been constructed successfully, which lays the foundation for further research on the genetically engineered vaccine against rotavirus. The novel secreted / transmembrane gene-modified vectors provide a powerful tool for studying post-synthesis processing and enhancing their immunogenicity.