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目的在前期筛选的HSV-2gD模拟抗原表位序列P6的基础上,在GenBank上选取与P6序列相对应且相似的HSV-2天然野生株gD的基因序列NP6,以pcDNA3.1(-)为载体、IL-18为佐剂,构建和表达NP6与IL-18串联的重组表达质粒,并观察免疫小鼠后的效果。方法采用串联简并引物PCR法构建重组质粒pcDNA3.1-IL18-NP6和pcDNA3.1-IL18。将构建的真核表达质粒pcDNA3.1-IL-18和pcDNA3.1-IL-18-HSVNP6肌内注射免疫BALB/c小鼠3次,每次间隔1周。末次免疫后1周眼眶静脉采血,ELISA法检测小鼠血清特异性抗体滴度、IFN-γ及IL-18含量。结果构建的串联重组质粒pcDNA3.1-IL18-NP6和pcDNA3.1-IL18经酶切及测序显示基因序列完全正确,间接免疫荧光、Western blot检测证实模拟抗原表位序列NP6具有近似天然序列的生物学活性;用表达质粒免疫小鼠,可刺激产生高滴度的特异性抗体,并可产生较高水平的IL-18和IFN-γ。结论成功构建和表达了重组质粒pcDNA3.1-IL18-NP6,表达产物具有免疫原性。
OBJECTIVE To select the sequence of HSV-2gD mimic epitope of P6 from pre-selected P6, and select the gene sequence of homologue and homology of gD of HSV-2 natural wild-type strain P6 corresponding to P6 sequence in GenBank. The sequence of pcDNA3.1 (-) was IL-18 was used as an adjuvant to construct and express the recombinant plasmid with NP6 and IL-18 in series, and the effect after immunization was observed. Methods Recombinant plasmids pcDNA3.1-IL18-NP6 and pcDNA3.1-IL18 were constructed by PCR using tandem degenerate primers. BALB / c mice were immunized intraperitoneally with the constructed eukaryotic expression plasmids pcDNA3.1-IL-18 and pcDNA3.1-IL-18-HSVNP6 three times at intervals of one week. Blood was collected from the orbital vein 1 week after the last immunization, and the serum specific antibody titers, IFN-γ and IL-18 levels were detected by ELISA. Results The constructed recombinant plasmids pcDNA3.1-IL18-NP6 and pcDNA3.1-IL18 were confirmed by restriction enzyme digestion and sequencing. The sequence of the gene was confirmed by indirect immunofluorescence and Western blot. Immunization of mice with expression plasmids stimulated the production of high titers of specific antibodies and produced higher levels of IL-18 and IFN-γ. Conclusion The recombinant plasmid pcDNA3.1-IL18-NP6 was successfully constructed and expressed. The expressed product was immunogenic.