论文部分内容阅读
预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。
Bprotein epitopes of the Vpr protein are predicted, and synthetic B-cell epitope peptides are used to prepare Vpr-specific antibodies. Bioinformatics techniques were used to obtain the consensus amino acid sequence of Vpr protein and to predict its potential B cell epitopes. The polypeptide was conjugated with carrier protein hemocyanin (KLH) and immunized rabbits to identify and purify the polypeptide-specific antibodies. Software prediction showed that the amino acid sequence of nucleotides 3 to 19 (N) and 82 to 95 (C) of the C terminus of Vpr protein were potential B cell epitopes. The titer of the polypeptide specific antibody in the antiserum was all detected by ELISA The results of Western-Blotting showed that both the N-terminal and the C-terminal of Vpr protein of HIV-1B or CRF07_BC recombinant could recognize specifically. Immunoprecipitation showed that Vpr peptides N and C Antibodies also specifically bind to the undenatured wild-type Vpr or GFP-Vpr fusion protein. The bioinformatics technology can successfully predict Vpr protein B cell epitopes, and the antibodies obtained by immunization have good specificity and applicability.