结核分枝杆菌CFP-10／ESAT-6融合蛋白模拟抗原表位的筛选

目的探讨噬菌体展示随机肽库技术在结核分枝杆菌培养滤过蛋白10（CFP-10）／早期分泌抗原靶点6（ESAT-6）融合蛋白（CE融合蛋白）模拟抗原表位筛选中的应用。方法以抗CE融合蛋白多克隆抗体为靶分子，对随机噬菌体7肽库进行筛选，经3轮生物淘选后，随机选取18个单噬菌体进行测序分析。采用双抗体夹心和竞争ELISA方法对测序后噬菌体进行阳性克隆及其活性鉴定。采用间接ELISA方法，选取阳性单噬菌体与CE融合蛋白分别对20份活动性肺结核患者和10份有卡介苗接种史健康人的血清标本抗体进行检测。结果经过3轮生物淘选，能与靶分子特异性结合的噬菌体得到了明显富集。18个单噬菌体测序共获得9种序列，其中单噬菌体5、6、18的氨基酸序列均包含Trp-Asp-Ala-Thr（WDAT）保守序列，该序列与ESAT-6第58-61位氨基酸的序列一致。9种序列中各取1个单噬菌体经双抗体夹心和竞争ELISA检测，有7个单噬菌体（1、5、6、10、13、14、18，S／N值依次为9.2、9.7、9.4、8.9、9.6、9.9、9.0）确定为具有免疫活性的阳性克隆。选取含有WDAT保守序列的阳性单噬菌体5与CE融合蛋白分别对2种血清标本抗体进行间接ELISA检测结果显示，单噬菌体5对2种血清标本抗体检测的吸光度值均高于CE融合蛋白（分别为0．931±0．298 vs 0．317±0．157、0．496±0．073 vs 0．118±0．026，均P〈0．05）；单噬菌体5对活动性肺结核患者血清标本抗体的检出率（95％，19／20）明显高于CE融合蛋白（60％，12／20），而对有卡介苗接种史健康人血清标本抗体的检出率（9／10）低于CE融合蛋白（10／10）。结论利用噬菌体展示随机肽库技术成功筛选出7个CE融合蛋白的模拟抗原表位，并获得了定位于ESAT-6第58-61位氨基酸序列的CE融合蛋白的1个线性B细胞抗原表位，提高了ELISA检测的敏感性，为进一步研究CE融合蛋白的?

Screening of mimotopes of CFP-10/ESAT-6 fusion protein from mycobacterium tuberculosis

Objective To screen the mimotopes of CFP-10/ESAT-6 (CE) fusion protein from mycobacterium tuberculosis using phage-displayed random peptide libraries. Methods The monoclonal antibody against the CE fusion protein was used to screen the binding peptide from the Ph.D.-7 peptide library. After 3 rounds of screening, 18 clones were randomly selected for DNA sequencing. The selected clones were tested by sandwich enzyme linked immunosorbent ELISA and competition ELISA. By indirect ELISA method, antibodies against single-chain phage positive clones and CE fusion protein were detected in serum samples from 20 patients with tuberculosis and 10 healthy people who had been vaccinated with BCG. Results After three rounds of biopanning, remarkable enrichment of the phage that can specifically bind with target molecules were observed.By DNA screening, 9 sequences were obtained, in which the sequences of single-chain phages 1, 7, 11, 12, 15, and 16 were exactly the same, and the conservative sequence Trp-Asp-Ala-Thr (WDAT), which was identical with the sequence from the 58th to 61st amino acids in ESAT-6, was found within the amino acid sequences of phages 5, 6, and 18. Nine phages with different sequences were detected using sandwich enzyme linked immunosorbent ELISA and competition ELISA, and 7 of them were found to be positive clones with immunoactivity (the S/N value of the phages 1, 5, 6, 10,13, 14, and 18 were 9.2, 9.7, 9.4, 8.9, 9.6, 9.9, and 9.0, respectively). Indirect ELISA using the positive phage 5 that containing WDAT sequence or CE fusion protein showed that the absorbance value of both the serum samples detected by using phage 5 were higher than that by CE fusion protein (0.931 ± 0.298 vs. 0.317 ± 0.157, 0.496 ± 0.073 vs. 0.118 ± 0.026, all P < 0.05). The detection rate of the serum samples from patients with tuberculosis by using phage 5 (95%, 19/20) that was significantly higher than that by using CE fusion protein (60%, 12/20), but the detection rate of the serum samples from healthy people was 9/10, which was lower than that by using CE fusion protein (10/10). Conclustions By using phage-displayed random peptide libraries, we screened out 7 mimotopes of CE fusion protein, and a liner B cell epitope positioned at the sequence of the 58th to 61st amino acids at ESAT-6. Basing on the results, the sensitivity of the ELISA test can be increased and detection reagent for anti-tuberculosis antibodies may be developed.