犬种布鲁氏菌ZG株单克隆抗体4H3抗原模拟表位的筛选及分析

Screening and analysis of mimic epitopes of monoclonal antibody 4H3 against Brucella canis ZG strain

摘要:

【背景】目前犬布鲁氏菌病诊断存在一定的困难。【目的】筛选并研究犬种布鲁氏菌单克隆抗体4H3株的特异性抗原表位。【方法】利用噬菌体肽库展示技术，以犬种布鲁氏菌单克隆抗体4H3株作为靶分子，包被酶标板，用12肽随机肽库经过3轮生物淘洗程序进行筛选。经过3轮筛选后，噬菌体产出率从5.00×10^-7增加到9.84×10^-6，假阳性率逐轮降低。从第3轮筛选的阳性克隆中随机挑取14个进行增殖，提取基因组DNA，进行测序分析；并通过iELISA和cELISA检测阳性克隆的亲和性和特异性。【结果】14株阳性单克隆噬菌体共出现3种不同的短肽序列，分别是KMSIRHPIRLPI、ILRRRRKRIIQI和QRIHMRLTTQS；iELISA结果表明3种短肽序列与单克隆抗体的亲和性依次为KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS；cELISA结果显示短肽KMSIRHPIRLPI和ILRRRRKRIIQI特异性较强。对亲和性较强、特异性较高的2条短肽KMSIRHPIRLPI和ILRRRRKRIIQI展开具体分析，比对分析表明KMSIRHPIRLPI与犬种布鲁氏菌RM6/66菌株的肽酶C26家族蛋白(PuuD)有比较高的相似性，氨基酸符合率为75%，而且存在4位连续相似氨基酸；ILRRRRKRIIQI与布鲁氏菌6/66菌株中3-氧酰基-[酰基载体蛋白]还原酶(OAR)相似性较高，氨基酸符合率为75%，存在2位连续相似氨基酸。【结论】利用噬菌体肽库展示技术筛选出与犬种布鲁氏菌ZG分离株单克隆抗体特异性结合的短肽序列。

Abstract:

[background] At present, there are some difficulties in the diagnosis of Brucella canis. [Objective] To screen and analyze the specific epitopes of monoclonal antibody 4H3 against Brucella canis. [Methods] The phage display peptide library was used for screening. The 4H3 against B. canis was used as the target molecule, which was employed to coat the ELISA plate. Then, we applied 12-mer phage display peptide library for screening through 3 rounds of biopanning. In the biopanning, yield of phage increased from 5.00×10^-7 to 9.84×10^-6, and the false positive rate gradually decreased. We selected 14 of the screened positive clones for amplification, followed by extraction and sequencing of the genomic DNA. The affinity and specificity of the positive clones were detected by iELISA and cELISA. [Results] There were 3 different kinds of short peptide sequences in 14 monoclonal phages, which were KMSIRHPIRLPI, ILRRRRKRIIQI, and QRIHMRLTTQS, respectively. The affinity of the 3 short peptide sequences to monoclonal antibodies was in the order of KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS, and KMSIRHPIRLPI and ILRRRRKRIIQI had strong specificity. Thus, we further analyzed KMSIRHPIRLPI and ILRRRRKRIIQI and found that KMSIRHPIRLPI showed high similarity to peptidase C26 family protein (PuuD) of B. canis RM6/66 (75% similarity in amino acids) with 4 consecutive similar amino acids. ILRRRRKRIIQI had high similarity to 3-oxoyl-[acyl carrier protein] reductase (OAR) of B. canis RM6/66 (75% similarity in amino acids), with 2 consecutive similar amino acids. [Conclusion] Based on phage display peptide library, the short peptide sequences specifically binding to the monoclonal antibody against B. canis ZG were screened out.