CAJ | 학술논문

目的构建人偏肺病毒(hMPV)DNA疫苗,小鼠免疫后评价其细胞和体液免疫水平.方法利用PCR方法,从hMPV的cDNA中扩增融合蛋白FATM(缺失跨膜区)基因和基质蛋白M基因,构建DNA疫苗pcDNA3.1His-FATM和pcDNA3.1His-M,瞬时转染后用Western Blot和间接免疫荧光方法检测F、M蛋白表达.疫苗肌内注射免疫小鼠,ELISA和ELISPOT方法分别检测血清IgG抗体和小鼠脾细胞CTL水平.结果 Western Blot和间接免疫荧光(IFA)方法证明构建的疫苗可表达FATM和M蛋白.peDNA3.1His-FATM单独免疫小鼠,血清抗体滴度为1:44;与pcDNA3.1His-M联合免疫后,血清抗体滴度为1:64.ELISPOT检测证明,联合免疫组小鼠脾细胞产生IFN-γ的效应CD8+T细胞数为42±8.9,高于单独免疫组32±7.4的水平.结论 DNA疫苗peDNA3.1His-F△TM可以诱导产生特异性的体液和细胞免疫,与pcDNA3.1His-M联合免疫,可以提高免疫水平.
목적 구건인편폐병독(hMPV)DNA역묘,소서면역후평개기세포화체액면역수평.방법 이용PCR방법,종hMPV적cDNA중확증융합단백FATM(결실과막구)기인화기질단백M기인,구건DNA역묘pcDNA3.1His-FATM화pcDNA3.1His-M,순시전염후용Western Blot화간접면역형광방법검측F、M단백표체.역묘기내주사면역소서,ELISA화ELISPOT방법분별검측혈청IgG항체화소서비세포CTL수평.결과 Western Blot화간접면역형광(IFA)방법증명구건적역묘가표체FATM화M단백.peDNA3.1His-FATM단독면역소서,혈청항체적도위1:44;여pcDNA3.1His-M연합면역후,혈청항체적도위1:64.ELISPOT검측증명,연합면역조소서비세포산생IFN-γ적효응CD8+T세포수위42±8.9,고우단독면역조32±7.4적수평.결론 DNA역묘peDNA3.1His-F△TM가이유도산생특이성적체액화세포면역,여pcDNA3.1His-M연합면역,가이제고면역수평.
Objective To construct human metapneumovirns (hMPV) DNA vaccines and evaluate the cellular and humoral immune response in mice.Methods Fusion protein FATM (without transmembrane domain) gene and M gene of hMPV were amplified from cDNA by PCR, then DNA vaccines pcDNA3. 1His-FΔTM and peDNA3.1His-M were constructed to verify the expression of F and M protein by Western blotting and indirect immunofluorescent assay (IFA) respectively. Serum IgG and spleen cell CTL were detected with ELISA and ELISPOT assay after the BALB/c mice were immunized intramuscularly with the vaccines. Results The candidate DNA vaccines could express FATM and M protein as detected with Western blotting and IFA. The IgG antibody titers of mice was 1:44 when immunized with poDNA3.1His-FΔTM, but could increase te 1:64 when co-immunized with pcDNA3.1His-M. ELISPOT assay demonstrated that IFN-γ-secreting effector T cells reached 42 ±8.9 in co-immunization group, higher than single vaccine pcDNA3.1His-FΔTM group (32 ± 7.4). Conclusion DNA vaccine pcDNA3.1His-FΔTM could induce specific cellular and humoral immune responses, and the immune response could increase when co-immunization with pcDNA3.1His-M was carried out.