重组质粒pcDNA3.1(-)-P6-gBCTL-TBK-1的免疫效果评价

Immunogenicity of the eukaryotic expression vector pcDNA3.1(-)-P6-gBCTL-TBK-1

目的观察以TBK-1为分子佐剂的重组质粒pc(pcDNA3.1-)-P6-g BCTL-TBK-1的免疫效果。方法将构建的真核表达质粒pc-P6-g BCTL-TBK-1转染HeLa细胞瞬时表达,Western blotting验证融合蛋白的表达。将重组质粒肌内注射免疫接种BALB/c小鼠,分别于第0、2、4周免疫接种3次,第5周采集免疫小鼠的眼眶静脉血,第8周处死小鼠后无菌分离小鼠脾脏。ELISA法检测免疫小鼠血清的特异性抗HSV-2g D-P6抗体滴度、γ干扰素(IFN-γ)、白细胞介素-18(IL-18)、白细胞介素-4(IL-4)和白细胞介素-6(IL-6)的浓度,MTT法检测脾淋巴细胞的增殖率,乳酸脱氢酶(LDH)法检测细胞毒性T淋巴细胞(CTL)的活性。结果以TANK binding kinase-1(TBK-1)为分子佐剂的重组质粒可刺激机体产生特异性的抗HSV-2g D-P6抗体,诱导分泌较高水平的IFN-γ、IL-4和IL-18,并能促进小鼠脾淋巴细胞的增殖和增强CTL活性。结论构建的HSV-2DNA疫苗pc-P6-g BCTL-TBK-1具有一定的免疫原性,能诱导产生一定强度的细胞免疫和体液免疫,TBK-1具有良好的分子伴侣作用。

Objective To investigate the immune effects of herpes simplex virus type 2（HSV-2） DNA vaccine of pc（pcDNA3.1-）-P6-g BCTL-TBK-1 in BALB/c mice.Methods The eukaryotic expression plasmid of pc-P6-g BCTLTBK-1 was constructed and transfected into He La cells,and the expressed protein was verified by Western blotting.The BALB/c mice were immunized by the plasmids through intramuscular injection for three times,in week 0,2 and 4.In week 5,the levels of interferon（IFN-γ）,interleukin-18（IL-18）,interleukin-4（IL-4）,interleukin-6（IL-6） and specific HSV-2g D-P6 IgG antibody were measured by ELISA.In week 8,spleen cells of vaccinated mice were separated and the proliferation of spleen lymphocytes were detected by M TT.The activity of cytotoxic T lymphocyte（CTL） was detected by lactic dehydrogenase（LDH） method.Results After being inoculated by the plasmid of pc-P6-g BCTL-TBK-1,the mice were induced to generate higher level of specific antibody,including IFN-γ,IL-18 and IL-4;the CTL kill cells＇ activity and proliferation capability of splenic lymphocytes were improved.Conclusion The recombinant plasmid of pc-P6-g BCTL-TBK-1 has immunogenicity,which can induce the humoral and cellular immune responses,and TBK-1may have good function of molecular chaperone.