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构建了含有狂犬病毒(RV)CVS株糖蛋白(GP)基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒(DNA疫苗)及空白载体质粒(对照组)免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义(P<0.01);不同的启动子(CMV或SV40)与不同GP基因(来源于CVS株或ERA株)对DNA疫苗的免疫效果无明显影响。在注射120d后.用PCR方法仍可检测出RVGP基因。结果表明:狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。
The recombinant plasmid pCMVCVSRG containing the glycoprotein (GP) gene of rabies virus (CV) strain was constructed and transfected into NIH3T3 cells. Both indirect immunofluorescence assay and APAAP assay showed that RVGP could be expressed in eukaryotic cells. The plasmids (DNA vaccine) and blank vector plasmid (control group) of the GP gene of different RV strains were respectively immunized with the mice, and the mice immunized with the DNA vaccine alone gave neutralizing antibodies. The survival rate of mice immunized with DNA vaccine was significantly higher than that of the control group after RV challenge (P <0.01). Different promoters (CMV or SV40) and different GP genes (derived from CVS strain or ERA strain) had no significant effect on the immunization effect of DNA vaccine. After 120 days of injection. RVGP gene can still be detected by PCR method. The results showed that rabies DNA vaccine induced low levels of neutralizing antibodies and memory B lymphocytes and protected mice against RV challenge. The vaccine is stable in the body. The rabies DNA vaccine developed for rabies immunization opened up a new way and laid the foundation for the development of DNA vaccine against other diseases.