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目的观察马来丝虫肌球蛋白29(Brugia malayi myosin 29,Bm M29)表位基因真核表达质粒pc DNA3.1(+)-Bm M29和原核表达的纯化重组蛋白r Bm M29免疫小鼠诱导的免疫应答效果。方法在大肠埃希菌(Escherichia coli)BL21诱导表达重组蛋白r Bm M29,纯化后作为重组蛋白疫苗;纯化真核表达重组质粒pc DNA3.1(+)-Bm M29作为核酸疫苗。核酸疫苗免疫注射部位为小鼠后腿胫前肌,重组蛋白为皮下多点注射。60只BALB/c小鼠随机分为A、B、C、D、E组,每组12只,分别注射PBS(100μg)、pc DNA3.1(+)/Cp G(100μg/30μg)、pc DNA3.1(+)-Bm M29/Cp G(100μg/30μg)、r Bm M29/Cp G(50μg/30μg)、pc DNA3.1(+)-Bm M29/r Bm M29/Cp G(前2次注射pc DNA3.1(+)-Bm M29/Cp G 100μg/30μg,第3次注射r Bm M29/Cp G 50μg/30μg),共免疫3次,每次免疫间隔2周。初次免疫后第4、6、8周,眼球摘除法采血制备血清,ELISA法检测免疫小鼠血清特异性Ig G抗体效价。免疫第8周处死小鼠,制备脾细胞悬液培养48 h,ELISA检测培养上清中细胞因子γ干扰素(IFN-γ)、白细胞介素-4(IL-4)水平。结果 ELISA检测结果显示,A、B、C、D和E组小鼠初次免疫后第4周血清中抗体的吸光度(A490值)分别为0.038±0.050、0.053±0.009、0.360±0.035、0.456±0.025、0.370±0.025,第6周分别为0.045±0.003、0.045±0.005、0.510±0.018、0.548±0.010、0.552±0.018,第8周分别为0.041±0.004、0.044±0.009、0.606±0.047、0.674±0.042、0.770±0.041,C、D、E组均高于A、B组(P<0.05),第8周时,E组Ig G抗体的A490值高于C组和D组(P<0.05)。初次免疫后第8周,A至E组小鼠脾细胞培养上清中IFN-γ的水平分别为(47.72±8.94)、(50.43±2.81)、(304.78±8.42)、(242.28±5.99)、(426.52±6.76)pg/ml,C、D、E组均高于A、B组(P<0.05),E组高于C组和D组(P<0.05);小鼠脾细胞培养上清中IL-4的水平分别为(60.00±11.14)、(57.71±15.95)、(93.17±12.56)、(96.67±11.48)、(101.17±5.81)pg/ml,C、D、E组均高于A、B组(P<0.05)。结论 pc DNA3.1(+)-Bm M29核酸疫苗和r Bm M29重组蛋白均可诱导BALB/c小鼠产生特异性体液和细胞免疫应答,核酸疫苗与重组蛋白疫苗联合免疫效果更佳。
Objective To observe the eukaryotic expression plasmid pcDNA3.1 (+) - Bm M29 of Brugia malayi myosin 29 (Bm M29) epitope and the recombinant protein rBm M29 induced by prokaryotic expression in mice Immune response effect. Methods The recombinant protein rBm M29 was induced in Escherichia coli BL21 and purified as a recombinant protein vaccine. The recombinant plasmid pcDNA3.1 (+) - Bm M29 was purified and expressed as a DNA vaccine. Nucleic acid vaccine immunization site for the hind anterior tibialis muscle, recombinant protein subcutaneous injection. Sixty BALB / c mice were randomly divided into A, B, C, D and E groups. Twelve mice in each group were injected with PBS (100μg), pcDNA3.1 (+) / CpG PcDNA3.1 (+) - Bm M29 / CpG (100 μg / 30 μg), rBm M29 / CpG (50 μg / 30 μg), pcDNA3.1 (+) - Bm M29 / rBm M29 / CpG Immunization was repeated 3 times with 2 weeks between immunizations with pcDNA3.1 (+) - Bm M29 / Cp G 100 μg / 30 μg and rBm M29 / Cp G 50 μg / 30 μg). At the 4th, 6th and 8th week after the primary immunization, serum was collected by eyeball exclusion method, and the titer of serum-specific Ig G antibody was detected by ELISA. Mice were sacrificed at 8 weeks after immunization. Spleen cell suspension was prepared and cultured for 48 h. The levels of IFN-γ and IL-4 in the culture supernatants were detected by ELISA. Results The results of ELISA showed that the absorbance (A490 value) of antibody in the 4th week after primary immunization in mice of groups A, B, C, D and E were 0.038 ± 0.050,0.053 ± 0.009,0.360 ± 0.035,0.456 ± 0.025 , 0.370 ± 0.025 in the sixth week, respectively, 0.045 ± 0.003, 0.045 ± 0.005, 0.510 ± 0.018, 0.548 ± 0.010, 0.552 ± 0.018 in the sixth week, 0.041 ± 0.004, 0.044 ± 0.009, 0.606 ± 0.047, 0.674 ± 0.042 , 0.770 ± 0.041, C, D and E were higher than those in A and B groups (P <0.05). At week 8, the A490 value of Ig G antibody in group E was higher than those in group C and D (P <0.05). At the 8th week after the first immunization, the levels of IFN-γ in the spleen cell culture supernatants of groups A to E were (47.72 ± 8.94), (50.43 ± 2.81), (304.78 ± 8.42), (242.28 ± 5.99), (426.52 ± 6.76) pg / ml, C, D and E were higher than those in A and B groups (P <0.05), and those in E group were higher than those in C and D groups The levels of IL-4 were (60.00 ± 11.14), (57.71 ± 15.95), (93.17 ± 12.56), (96.67 ± 11.48), (101.17 ± 5.81) pg / ml in group C, A, B group (P <0.05). Conclusions Both pc DNA3.1 (+) - Bm M29 DNA vaccine and r Bm M29 recombinant protein can induce specific humoral and cellular immune responses in BALB / c mice. The combination of DNA vaccine and recombinant protein vaccine is more effective.