TIMP-1 B细胞表位的预测和鉴定

Prediction and identification of B-cell epitopes of TIMP-1

目的:预测并鉴定金属蛋白酶组织抑制物-1(TIMP-1)蛋白B细胞表位。方法:采用DNAStar和BcePred分析软件联合预测TIMP-1的B细胞表位,以此合成8分支多抗原肽结构的表位肽(MAP),并与通用型T辅助表位肽(VQGEESNDK,氨基酸163～171)联合免疫家兔,检测免疫血清效价,用Western blotting和间接酶联免疫吸附测定等方法鉴定其特异性和抗体亲和力。结果:软件预测显示,TIMP-1的第27～41位(MAP1)、第57～71位(MAP2)、第95～109位(MAP3)和第193～207位(MAP4)氨基酸序列最可能为其优势B细胞表位。抗体滴度动态检测表明,MAP2、MAP3和MAP4均能诱导产生特异性抗体,其中MAP2和MAP4诱导的抗体水平最高;免疫印迹证实MAP2、MAP3和MAP4诱导产生特异性的TIMP-1抗体;间接ELISA证实MAP4与商品化TIMP-1抗体具有最高的亲和力。结论:TIMP-1的第27～41位和第193～207位氨基酸为其优势B细胞表位,其中第193～207位氨基酸的免疫原性最强,这为TIMP-1多肽抗体和B细胞优势短肽疫苗研制提供了理论依据。

AIM ： To predict and identify the B - cell epitopes of human tissue inhibitor of metalloproteinase - 1 （ TIMP - 1 ）. METHODS： The prediction of B - cell epitopes of TIMP - 1 was conducted by I）NAStar and BcePred soft-wares, and 8 -branch form of multiple antigen peptide （MAP） was synthesized, which were subsequently used for immuni- zing rabbits mixed with an universal T -helper epitope human IL - 1β peptide （VQGEESNDK, amino acid 163 - 171 ）. The immunogenicity of the synthesized peptide was evaluated by monitoring the antiserum titer and analysis of binding affinity with antibody. The specificity of produced antiserum was detected by Western blotting. RESULTS： Amino acids 27 - 41 （ MAP1 ）, 57 - 71 （MAP2） ,95 - 109 （MAP3） and 193 - 207 （MAP4） of TIMP - 1 were predicted as the most potential epitopes and synthesized as immunogen. MAP2, MAP3 and MAP4 induced antibodies with high titer by the dynamic monitoring, which was also identified as specific antibody against TIMP - 1 by Western blotting. Affinity analysis showed MAP4 had the highest binding activity with market antibody. CONCLUSION： The No. 57 ~ 71 and 193 -207 peptides of TIMP - 1 protein are proved to be the preponderant B - cell epitopes and the 193 ~ 207 epitope is believed to have the strongest immunogenicity, which may assist the development of therapeutic approach against high expression of TIMP - 1.