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目的:通过培养和扩增Ⅱ型单纯疱疹病毒(HSV-2)并提取其全基因组,从而克隆包膜糖蛋白gG-2全长基因及其保守性较高、抗原性较强的特异片段。方法:将HSV-2病毒感染Hela细胞获得大量病毒液,用酚-氯仿法提取病毒全长基因组,并以此为模板扩增编码gG-2的US4基因,再根据氨基酸序列比对结果,选取其特异片段,设计带酶切位点的引物克隆gG-2基因特异片段。结果:成功地获得了大量HSV-2病毒液并提取了病毒基因组,克隆了gG-2全长基因以及特异片段,测序证实,两者均与GenBank中报道的序列相一致。结论:对于遗传特性相对稳定的HSV-2来说,通过病毒感染细胞的方法短时期内获得大量病毒液,并从病毒全基因组中克隆gG-2特异基因片段的方法是可行的,从而为进一步构建gG-2特异片段相关载体、表达相应蛋白奠定了基础。
OBJECTIVE: To clone the full-length gG-2 gene of envelope glycoprotein and to find a more conservative and antigen-specific fragment by culturing and amplifying herpes simplex virus type 2 (HSV-2) and extracting its whole genome. Methods: A large amount of virus solution was obtained from Hela cells infected with HSV-2 virus. The full-length genome was extracted by phenol-chloroform method and used as a template to amplify the US4 gene encoding gG-2. Based on the amino acid sequence alignment results, Its specific fragment, primers designed with restriction sites cloned gG-2 gene-specific fragments. Results: A large number of HSV-2 virus fluids were successfully obtained and the viral genome was extracted. The full-length gG-2 gene and specific fragments were cloned and confirmed by sequencing. Both of them were consistent with the sequences reported in GenBank. CONCLUSION: For HSV-2 with relatively stable genetic characteristics, it is feasible to obtain a large amount of virus solution in a short period of time by virus-infected cells and to clone the gG-2 specific gene fragment from the whole genome of the virus, The construction of gG-2 specific fragment related vectors, the expression of the corresponding protein laid the foundation.