重组屋尘螨变应原突变体的免疫原性与过敏原性研究

Immunogenicity and allergenicityof recombinant Der p2 mutant allergen

目的 构建屋尘螨Der p2变应原突变体的原核表达质粒,并在大肠杆菌中表达、纯化,以观察屋尘螨Der p2变应原突变体的免疫原性以及抗原性.方法 基因敲除屋尘螨Der p2变应原的部分氨基酸残基序列,将编码基因定向克隆到pET32a原核表达质粒中,转入BL21表达菌中并提纯;将18只Balb/c小鼠随机分为Der p2变应原突变体组、重组Der p2组和对照组.采用酶联免疫吸附试验（ELISA）测定各组小鼠血清中IgE、IgG1与IgG2a抗体变化,用免疫斑点试验测定Der p2突变体的IgE结合能力.结果 成功构建了Der p2变应原突变体的原核表达质粒,表达并纯化了Der p2变应原突变体.腹腔免疫后0d,各组IgE、IgG1与IgG2a抗体表达无明显变化（P=0.067、P=0.052、P=0.078）;免疫后第7d,重组Der p2变应原组诱导IgE抗体的产生量高于Der p2变应原突变体组（P =0.002）,重组Der p2变应原组诱导的IgG2a抗体高于Der p2变应原突变体组（P=0.0056）;腹腔免疫后第14 d,重组Der p2变应原组IgE抗体的产生量高于Der p2变应原突变体组（P=0.001）,Der p2变应原突变体组IgG2a低于重组Der p2变应原组（P=0.0034）.Der p2变应原突变体与屋尘螨过敏患者的阳性血清结合明显较低.结论 Der p2变应原突变体能诱导小鼠产生IgG1抗体和IgG2a抗体,同时能明显减少IgE抗体的产生,并且Der p2变应原突变体具有较低的IgE结合能力.

Objective To observe the immunogenicity and allergenicity of recombinant house dust mite Der p2 allergen （rDer p2） mutants.Methods Der p2＆#39;s IgE epitope was deleted.The DNA encoding Der p2 mutant were synthesized and cloned into pET32a plasmid.rDer p2 mutant were expressed in E.coli（BL21） and purified through Ni ＋ NTA.Balb/c mice were sensitized by rDer p2 or rDer p2 mutant.The levels of IgE,IgG1,and IgG2a antibody in serum were determinate by enzyme-linked immunosorbent assay （ELISA）.The binding ability of rDer p2 mutant with IgE was measured by dot immunobirding assay.Results rDer p2 mutant was expressed and purified.The first day after immunization,the levels of IgG1,IgG2a,and IgE antibody had no obvious difference between rDer p2 and rDer p2 mutant group（P =0.067,P=0.052,P=0.078）..After 7 days of immunization,the levels of IgG1 antibody had no significant difference between rDer p2 and rDer p2 mutant （P=0.0612）.However,the level of IgE antibody in rDer p2 mutant group was lower than rDer p2 （P=0.002） group.The level of IgG2a antibody in rDer p2 mutant group was lower than rDer p2 group （P =0.0056）.After 14 days,the level of IgE antibody in rDer p2mutant group was much lower than rDer p2 group （P =0.001）.The level of IgG2a antibody in rDer p2 mutant group was much lower than rDer p2group （P =0.0034）.Compared with rDer p2,rDer p2 mutant had the weak binding ability with house dust mites allergic patients.Conclusion rDer p2 mutant could induce mice to produce IgG2a,IgG1 antibody and to reduce IgE antibody production at the same time.rDer p2 mutant exhibited best immunogenicity and lower allergenicity.