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

发布时间：2018-03-18 20:31

本文选题：马来丝虫　切入点：肌球蛋白表位基因　出处：《中国寄生虫学与寄生虫病杂志》2017年01期 　论文类型：期刊论文

【摘要】：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(P0.05),第8周时,E组Ig G抗体的A490值高于C组和D组(P0.05)。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(P0.05),E组高于C组和D组(P0.05);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(P0.05)。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。
[Abstract]:Objective to observe the immune response induced by eukaryotic expression plasmid pc DNA3.1 (pc BmM29) of myosin 29 Brugia malayi myosin 29 (BmM29) and recombinant protein rBmM29 of E. coli Escherichia coli. Methods the recombinant protein rBmM29 was used to immunize mice with the recombinant protein rBmM29. Methods the recombinant protein rBmM29 was used to immunize mice with Escherichia coli)BL21. The recombinant protein rBmM29 was induced to express and purified as a recombinant protein vaccine. The recombinant eukaryotic expression plasmid pc DNA3.1 was purified. The injection site of nucleic acid vaccine was the anterior tibial muscle of the hind leg of mice, and the recombinant protein was injected subcutaneously. 60 BALB/c mice were randomly divided into 2 groups, 12 in each group. Pc DNA 3.1 (Bm M29r / Bm M29Cp / 100 渭 g / 30 渭 g / 30 渭 g / 100 渭 g / 30 渭 g / 100 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / 30 渭 g / g, 50 渭 g / 30 渭 g / g, respectively) (Bm / M29r / Bm M29cp / 100 渭 g / 30 渭 g / g of pc DNA) (Bm M29r / M 29Cp / 100 渭 g / g / 30 渭 g / g, third injection of r Bm M29Cp / G 50 渭 g / 30 渭 g / g / 30 渭 g / g), 2 weeks after first immunization, 468 weeks after first immunization, 4 渭 g / g / 30 渭 g / g of pc DNA (Bm M29r / Bm M29Cp / 100 渭 g / 30 渭 g / g) respectively. Serum prepared by enucleation of eyeball was used to detect the specific IgG antibody titer of immunized mice by Elisa. The mice were killed at the 8th week after immunization. The levels of IFN- 纬 and IL-4 in the supernatant were detected by Elisa. Results the results of ELISA showed that the absorbance of the antibody in serum of the mice in group E and group A was 4 weeks after the first immunization, and the results of Elisa showed that the levels of IFN- 纬 and IL-4 in the supernatant of the supernatant were determined by Elisa. Results the results of ELISA showed that the antibody in the serum of the mice in the first immunization group was A490. The values were 0.038 卤0.050, 0.053 卤0.009, 0.360 卤0.035, 0.456 卤0.025, 0.370 卤0.025, 0.045 卤0.0030.045 卤0.005, 0.510 卤0.018, 0.548 卤0.010, 0.552 卤0.018, 0.041 卤0.0040.044 卤0.009, 0.606 卤0.047, 0.674 卤0.0420.770 卤0.0420.7 卤0.041, respectively. At the 8th week, the A490 value of IgG antibody in E group was higher than that in C group and D group. 8 weeks after primary immunization, the A490 value of IgG antibody in E group was higher than that in E group. The levels of IFN- 纬 in the culture supernatant of mice were 47.72 卤8.94, 50.43 卤2.81, 304.78 卤8.42, 242.28 卤5.9921, 426.52 卤6.76pgml / ml, 426.52 卤6.76, respectively, and the levels of IL-4 in the culture supernatant of spleen cells in mice were higher than those in group A and B, respectively. Conclusion the levels of IL-4 in the culture supernatant of spleen cells of mice were 60.00 卤11.147.71 卤15.957.71 卤15.957.17 卤11.48A, respectively. Induce specific humoral and cellular immune responses in BALB/c mice. The combined immunization effect of nucleic acid vaccine and recombinant protein vaccine is better.
【作者单位】：南通大学医学院病原生物学系;
【基金】：南通市应用研究计划项目(No.MS12015115) 南通大学大学生创新训练计划项目(No.yxy201509)~~
【分类号】：R392

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