不同复性方法纯化的重组大肠杆菌外膜蛋白A小鼠免疫原性研究  

Immunogenicity of Recombinant Escherichia coli Outer Membrane Protein A(rOmpA)Purified by Different Renatured Methods in Mice

作　　者：肖雪 杨思宇 翟璐 夏洪伟 高玉龙 吴金 张中洋 朱战波[1] 崔玉东[1] Xiao Xue;Yang Siyu;Zhai Lu;Xia Hongwei;Gao Yulong;Wu Jin;Zhang Zhongyang;Zhu Zhanbo;Cui Yudong(Heilongjiang Bayi Agricultural University,Daqing163319;Heyuan Bioengineering Co.,LTD)

机构地区：[1]黑龙江八一农垦大学,大庆163319 [2]和元生物工程股份有限公司

出　　处：《黑龙江八一农垦大学学报》2021年第1期68-75,共8页journal of heilongjiang bayi agricultural university

摘　　要：为探究不同复性方法制备的重组大肠杆菌外膜蛋白A(rOmpA)的免疫原性,研究分别用纯化的β-环糊精复性rOmpA(β-rOmpA)、吐温-20复性rOmpA(T-rOmpA)和包涵体形式rOmpA(I-rOmpA)作为抗原,与弗氏佐剂乳化后肌肉注射免疫接种小鼠。通过检测血清特异性抗体、脾淋巴细胞分泌细胞因子及攻毒试验进行免疫效力评价。结果,β-rOmpA免疫组小鼠血清抗体水平高于T-rOmpA免疫组和I-rOmpA免疫组(P<0.05);三种抗原免疫均能提高小鼠淋巴细胞分泌IFN-γ水平,且在体外由rOmpA刺激下,β-rOmpA免疫组小鼠产生的IFN-γ、IL-17A水平显著高于T-rOmpA和I-rOmpA免疫组(P<0.05);用E.coli 2002-1菌株腹腔攻毒后,β-rOmpA免疫组小鼠肝、脾、肺、肾中的细菌定植量显著低于T-rOmpA免疫组和I-rOmpA免疫组(P<0.001);攻毒后第2 d PBS对照组小鼠免疫保护率为0时,β-rOmpA免疫组为60%,T-rOmpA免疫组为40%,I-rOmpA免疫组为30%。结果表明,β-环糊精复性纯化的rOmpA免疫原性最好,诱导机体产生免疫应答和免疫保护作用最强。To evaluate the immunogenicity of E.coli outer membrane protein A(rOmpA)prepared by different renaturation methods,the purified rOmpA refolded byβ-cyclodextrin(β-rOmpA)and Tween 20(T-rOmpA)and rOmpA in inclusion body form(I-rOmpA)were used as antigen,and the mice were immunized by intramuscular injection after emulsification with Freund’s adjuvant.The immune efficacy was evaluated by detecting the serum antibody,the cytokines secreted by spleen lymphocytes and experimental challenge.The results showed that the serum antibody level of mice inβ-rOmpA immunized group was higher than that in T-rOmpA and I-rOmpA immunized group(P<0.05).The level of cytokine IFN-γwas increased in all the three immunized groups,and theβ-rOmpA immunized group produced significantly higher levels of IFN-γand IL-17A under the stimulation with rOmpA than T-rOmpA and I-rOmpA immunized group(P<0.05).After intraperitoneal challenge with E.coli 2002-1,the numbers of bacterial colonization in Liver,spleen,lung and kidney of mice immunized withβ-rOmpA were significantly lower than that of mice immunized with T-rOmpA or I-rOmpA(P<0.001).When all the mice in the PBS group died on the second day after the challenge,60%mice were protected inβ-rOmpA immunized group,40%mice were protected in T-rOmpA immunized group,30%mice were protected in I-rOmpA immunized group.The results showed thatβ-rOmpA could induce the body to produce a good immune response and immune protection.

关 键 词：埃希氏大肠杆菌 rOmpA Β-环糊精 复性 免疫原性 

分 类 号：Q78[生物学—分子生物学]