马来丝虫肌球蛋白29表位基因真核表达质粒及原核表达蛋白免疫小鼠诱导的免疫应答

Mouse immune responses elicited by eukaryotic expression plasmid and prokaryotically expressed protein of Brugia malayi myosin 29 epitope

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 determine the immune responses elicited in BALB/c mice by vaccination with eukaryotic expression plasmid pcDNA3.1（＋）-BmM29 containing Brugia malayi myosin 29（BmM29） epitode and prokaryotically expressed recombinant BmM2 protein（rBmM29） respectively. Methods rBmM29 protein was expressed in E. coli strain BL21, purified as recombinant protein vaccine, and administered via multiple subcutaneous injections. The purified recombinant plasmid pcDNA3.1（＋）-BmM29 was used as the nucleic acid vaccine and injected into the tibialis anterior muscle. Sixty BALB/c mice were randomized to receive three immunizations（with intervals of 2 weeks） with PBS （100 μg, group A）, pcDNA3.1（＋）/CpG （100 μg/30 μg, group B）, pcDNA3.1（＋）-BmM29/CpG （100 μg/30 μg, group C）, rBmM29/CpG（50 μg/30 μg, group D）, or pcDNA3.1（＋）-BmM29/rBmM29/CpG （two injections of pcDNA3.1（＋）-BmM29/CpG 100 μg/30 μg followed by a rBmM29/CpG 50 μg/30 μg）. Serum was prepared through ophthalmectomy at week 4, 6, and 8 after primary immunization, and the serum IgG titer was determined by ELISA. The mice were sacrificed at week 8, splenocyte suspension cultured for 48 h, and levels of INF-γ and IL-4 in the supernatant detected by ELISA. Results ELISA results showed that the A490 values of serum IgG in groups A-E were 0.038 ± 0.050,0.053 ± 0.009,0.360 ± 0.035,0.456 ± 0.025,0.370 ± 0.025 at week 4,0.045 ± 0.003,0.045 ± 0.005,0.510 ± 0.018,0.548 ± 0.010,0.552 ± 0.018 at week 6, and 0.041 ± 0.004,0.044 ± 0.009,0.606 ± 0.047,0.674 ± 0.042,0.770 ± 0.041 at week 8, significantly higher in groups C, D and E than in groups A and B （P 〈 0.05） at all time points, and significantly higher in group E than in groups C and D（P 〈 0.05） at week 8. The IFN-γ levels in splenocyte culture supernatant at week 8 after primary immunization were （47.72 ± 8.94）,（50.43 ± 2.81）,（304.78 ± 8.42）,（242.28 ± 5.99）, and（426.52 ± 6.76） pg/ml in groups A-E, respectively, significantly higher in groups C-D than in groups A and B（P 〈 0.05）, and in group E than in groups C and D（P 〈 0.05）. The IL-4 levels in splenocyte culture supernatant were（60.00 ± 11.14）,（57.71 ± 15.95）,（93.17 ± 12.56）,（96.67 ± 11.48）, and （101.17 ± 5.81） pg/ml, significantly higher in groups C-D than in groups A and B（P 〈 0.05）. Conclusion Both the recombinant plasmid pcDNA3.19（＋）-BmM29 and rBmM29 protein could elicit specific humoral and cellular immune responses in mice. Combined immunization with nucleic acid vaccine and protein vaccine is superior to each of the two alone.