摘要
目的:预测并鉴定沙眼衣原体(Ct)Tarp蛋白的B细胞表位,为进一步研究Tarp蛋白的生物学特性及表位疫苗研制奠定基础。方法:利用生物信息学软件综合分析、预测筛选E血清型Ct Tarp蛋白的B细胞表位,获得6个潜在目的表位。将人工合成的表位肽段与弗氏佐剂充分乳化,经背部皮下多点注射途径免疫BALB/c小鼠(每次100μg/只,3只/组,共24只),间隔2周,共免疫3次。采用ELISA法检测免疫小鼠血清中Tarp蛋白特异性抗体IgG及其亚类IgG1、IgG2a,以及阴道分泌物中Tarp蛋白特异性sIgA抗体;进一步采用间接免疫荧光实验、免疫沉淀实验和Western blot法检测表位免疫的血清抗体与Tarp蛋白结合的特异性。结果:筛选并鉴定了Tarp蛋白的一个免疫优势B细胞表位(aa171-186),该表位可以刺激小鼠产生较高水平的Tarp蛋白特异性IgG抗体和sIgA抗体,且抗体亚类以IgG1为主。免疫荧光实验、免疫沉淀实验和Western blot法检测结果显示,该段表位肽免疫血清抗体可特异性识别Tarp蛋白。结论:获得了Ct Tarp蛋白的一个免疫优势B细胞表位,为进一步研究Tarp蛋白的生物学特性及表位疫苗研制奠定基础。
Objective： To predict and identify the B-cell epitope ofChlamydia trachomatis Tarp.Methods：The amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and 6 possible linear B-cell epitopes peptides （aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513） with high predicted antigenicity and high conservation were investigated. After coupling to KLH, the syn-thetic peptides were emulsiifeld with Freund’s adjuvant. Then, BALB/c mice were randomly assigned to receive （subcutaneous injection） peptides-KLH, KLH or PBS （n=3, 100 μg/time/mouse）, respectively, and the same immunization schedule was repeated third times at a 2-week interval. ELISA was used to detect the IgG and sub-types IgG1, IgG2a in the serum as well as the sIgA in the vaginal secretions. Furthermore, the speciifc binding of anti-sera produced by epitopes immunization to natural Tarp antigen was detected with indirect immunolfuo-rescence assay （IFA）, immunoprecipitated and Western blot analysis, respectively.Results： The epitope peptide F4 （aa171-186） was able to induce signiifcant Tarp speciifc antibody IgG in serum and sIgA in vaginal wash as compared with KLH or PBS （1：100） （P〈0.05）, and the major IgG antibody subtype was IgG1. Western blot and IFA results conifrmed that speciifc antibodies were induced by epitope aa171-186 recognized natural Tarp anti-gen.Conclusion： Our results demonstrated that the predictive epitopes （aa171-186） are immunodominant B-cell epitope of Tarp, and if proven protective and safe, in conjunction with others well-documented epitopes, they may be included into a candidate epitope-based vaccine.
出处
《温州医学院学报》
CAS
2014年第6期395-400,共6页
Journal of Wenzhou Medical College
基金
浙江省自然科学基金资助项目(Y2100611)
温州市科技局科研基金资助项目(Y20100090
Y20110083)
关键词
沙眼衣原体
Tarp蛋白
B细胞表位
小鼠
Chlamydia trachomatis
translocated actin-recruiting phosphoprotein （Tarp）
B-cell epitope
mice
