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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,以pcDNA3.1(-)为载体、IL-18为佐剂,构建P6与IL-18串联的重组表达质粒,并在真核细胞中表达。将重组质粒免疫小鼠,观察免疫效果。方法:采用串联简并引物PCR法构建重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18,以酶切、测序鉴定。将重组质粒分别转染CHO细胞进行瞬时表达,通过间接免疫荧光和Western blot法检测P6的表达情况。将构建的真核表达质粒肌内注射免疫接种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;末次免疫后1个月,处死小鼠,无菌分离脾脏,MTT法测定脾淋巴细胞增殖率。结果:构建的串联重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18经酶切及测序显示基因序列完全正确,经间接免疫荧光、Western blot法检测可证实模拟抗原表位序列P6具有近似天然序列的生物学活性。重组核酸疫苗pcDNA3.1-IL-18-HSV P6免疫小鼠后可刺激血清特异性抗体的产生,诱导分泌较高水平的IFN-γ和IL-18。可促进小鼠脾淋巴细胞的增殖。结论:成功构建和表达了重组质粒pcD-NA3.1-IL18-P6,其能诱导较强的细胞免疫和体液免疫,从而为构建更加有效的HSV-2DNA疫苗奠定了良好的基础。
OBJECTIVE: To construct a recombinant plasmid containing P6 and IL-18 in tandem with pcDNA3.1 (-) as carrier and IL-18 as adjuvant on the basis of P6 sequence of HSV-2gD previously screened. Eukaryotic cells. The recombinant plasmid was immunized mice to observe the immune effect. Methods: Recombinant plasmids pcDNA3.1-IL18-P6 and pcDNA3.1-IL18 were constructed by PCR using tandem degenerate primers and identified by restriction enzyme digestion and sequencing. The recombinant plasmids were transfected into CHO cells for transient expression. The expression of P6 was detected by indirect immunofluorescence and Western blot. BALB / c mice were immunized intramuscularly with the constructed eukaryotic expression plasmids for 3 times at intervals of 1 week. Serum specific antibody titers, IFN-γ and IL-18 levels were measured by ELISA method in 1 week after the last immunization. One month after the last immunization, the mice were sacrificed and the spleens were aseptically isolated. The spleen was determined by MTT assay Lymphocyte proliferation rate. Results: The constructed recombinant plasmid pcDNA3.1-IL18-P6 and pcDNA3.1-IL18 by enzyme digestion and sequencing showed that the gene sequence was completely correct. Indirect immunofluorescence and Western blot confirmed that the pseudo-epitope sequence P6 was similar Biological activity of natural sequences. Recombinant DNA vaccine pcDNA3.1-IL-18-HSV P6 immunized mice stimulated the production of serum specific antibodies and induced the secretion of higher levels of IFN-γ and IL-18. Can promote mouse spleen lymphocyte proliferation. CONCLUSION: The recombinant plasmid pcD-NA3.1-IL18-P6 was successfully constructed and expressed, which can induce strong cellular and humoral immunity, which laid a good foundation for constructing a more effective HSV-2 DNA vaccine.