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目的:构建含两种狂犬病毒疫苗株的融合糖蛋白基因DNA疫苗并对小鼠的免疫效果进行评价。方法:用RT-PCR法分别扩增和分离出SRV9株和CTN株的狂犬病毒糖蛋白部分表位的基因(SRV9毒株的1~252氨基酸和CTN毒株253~524氨基酸),构建含融合G蛋白基因的DNA疫苗质粒VR1055-SRV9CTN。碱裂解法大量提取重组质粒并经Sepharose 4FF柱层析纯化后直接肌肉注射免疫NIH小鼠,用ELISA法和淋巴细胞转化实验分别检测质粒DNA疫苗诱导小鼠产生抗狂犬病毒的体液免疫和细胞免疫效果。CTLL-2依赖细胞株/MTT比色法进行IL-2活性测定。结果:VR1055-SRV9CTN DNA疫苗具有较好的诱生狂犬病毒抗体能力。诱导细胞免疫方面,质粒VR1055-SRV9CTN DNA疫苗和空载体对照组的ConA刺激指数存在显著性差异(t=7.201,P=0.000)。小鼠血清中的IL-2活性测定表明DNA疫苗组显著高于空载体组(t=21.616,P=0.000)。CVS株RV脑内攻击保护实验中,疫苗组和对照组小鼠存活率分别为63%、25%,二者间差异具有显著性(t=7.065,P=0.008)。结论:含狂犬病毒糖蛋白基因VR1055-SRV9CTN DNA疫苗既能诱导体液免疫又能诱导细胞免疫,具有较好的免疫保护效果。
OBJECTIVE: To construct a DNA vaccine of fusion glycoprotein gene containing two rabies virus vaccine strains and to evaluate the immune effect of mice. Methods: The partial epitopes of rabies virus glycoprotein from SRV9 strain and CTN strain (1-252 amino acids and 253-524 amino acids from CTN strain) of SRV9 strain were amplified and separated by RT-PCR respectively. G protein gene DNA vaccine plasmid VR1055-SRV9CTN. After alkaline lysis, a large amount of recombinant plasmids were extracted and purified by Sepharose 4 FF column chromatography. The NIH mice were immunized by intramuscular injection directly. ELISA and lymphocyte transformation were used to detect the anti-rabies virus humoral and cellular immunity effect. CTLL-2-dependent cell line / MTT colorimetric determination of IL-2 activity. Results: The VR1055-SRV9CTN DNA vaccine had good ability to induce rabies virus antibody. In the induction of cellular immunity, the ConA stimulation index of plasmid VR1055-SRV9CTN DNA vaccine and empty vector control group showed significant difference (t = 7.201, P = 0.000). Measurement of IL-2 activity in mouse serum showed that the DNA vaccine group was significantly higher than the empty vector group (t = 21.616, P = 0.000). CVS strain RV brain challenge protection experiments, the vaccine group and control group mice survival rates were 63%, 25%, the difference between the two was significant (t = 7.065, P = 0.008). Conclusion: The VR1055-SRV9CTN DNA vaccine containing rabies virus glycoprotein gene can induce both humoral and cellular immunity, and has good immunoprotective effect.