HSV-2gD核酸疫苗的构建及免疫效果观察

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。

Construction of a recombinant eukaryotic expression vector containing HSV 2gD and its immune responses

AIM: To construct and express pcDNA3.1-IL-18-HSV P6 recombinant DNA vaccine,and to observe the immune responses of pcDNA3.1-IL-18-HSV P6 genetic vaccine.METHODS: Genes of P6 and IL-18 were cloned into the eukaryotic expression vector pcDNA3.1(-) respectively,and confirmed by enzyme digestion and sequence analysis,the recombinant plasmid pcDNA3.1-IL-18-HSVP6 was transformed into CHO cells.The expressed protein was characterized by indirect immunofluorescence and Western blotting.The recombinant plasmid pcDNA3.1-IL-18-HSVP6 was used to inoculate BALB/c mice by intramuscular injection for three times,once a week.One week after the last vaccination,the levels of specific IgG antibody,IFN-γ and IL-18 were detected by ELISA;One month after the last vaccination,spleen cells of vaccinated mice were separated and proliferation of spleen lymphocytes were detected by MTT assay.RESULTS: Recombinant pcDNA3.1-IL-18-HSV P6 plasmid was confirmed by enzyme digestion and DNA sequencing.After inoculated by pcDNA3.1-IL-18-HSV P6 vaccine,the mice could produce higher level of splenocytes proliferation and secrected higher level of IFN-γ,IL-18 and specific antibody.CONCLUSION: DNA vaccine of pcDNA3.1-IL-18-HSV P6 has been successfully constructed,and it can effectively induce humoral and cellular immune responses,which prorided a basis for constructing new type of DNA vaccine.