结核分枝杆菌抗原模拟表位肽的筛选及免疫活性鉴定

目的：从噬菌体随机12肽库中筛选结核分枝杆菌模拟抗原表位，并初步研究其免疫活性。方法：以鹿结核阳性血清纯化多克隆抗体IgG作为固相配基筛选噬菌体随机12肽库，按吸附-洗脱-扩增过程进行3轮筛选，随机挑取20个单克隆进行特异性鉴定，将筛选后得到的阳性克隆扩增后免疫BALB/c小鼠作为实验组；以TBS为阴性对照组，BCG为阳性对照组，采用间接ELISA方法检测抗体效价，MTT法检测淋巴细胞增殖能力。每组小鼠免疫3周后再鼻腔接种BCG，3周后无菌取左全肺研磨计算肺脏荷菌量，取右全肺制备病理切片观察肺脏病理变化。结果：经三轮筛选后阳性克隆得到富集，ELISA鉴定20个单克隆中有15个阳性克隆阳性率为75%。免疫后实验组小鼠抗体水平高于BCG组(P<0.01)。小鼠脾淋巴细胞增殖实验，经ConA、LPS、PPD刺激后实验组SI值均高于BCG组，但差异无显著性(P>0.05)，与TBS组比较差异有显著性(P<0.01)。实验组、BCG组均观察到少量的淋巴细胞浸润，而TBS组肺泡结构消失，组织发生实变。肺脏荷菌量实验显示实验组肺脏荷菌量［(4.080±0.035)log CFU·mg^-1)］ 低于TBS组［（4.360±0.110)log CFU·mg^-1］　(P<0.01)，但高于BCG组荷菌量［(3.980±0.023)log CFU·mg^-1］(P>0.05)。结论：成功筛选得到含有结核分枝杆菌抗原模拟表位的阳性克隆，这些阳性克隆能有效诱导小鼠产生比BCG更高的抗体效价和保护效应，对其进行进一步序列测定和功能分析将有助于结核病疫苗的开发。

Screening of Mycobacterium tuberculosis mimotopes from phage display peptide library and identification of immune activity

Objective To screen mimic peptide epitopes of Mycobacterium tuberculosis from 12-mer phage display random peptide library,and to explore its immune activity.Methods A 12-mer phage display random peptide library was screened for 3 rounds by using the polyclonal antibody IgG which was purified from tubercle positive serum of deers according to such a procedure as adsorbing,eluting and amplification,and 20 positive clones were selected randomly,confirmed by its specificity.The BALB/c mice were immuned with mix...