结核分枝杆菌抗原模拟表位肽的筛选及免疫活性鉴定

J4 ›› 2009, Vol. 35 ›› Issue (4): 664-668.

结核分枝杆菌抗原模拟表位肽的筛选及免疫活性鉴定

张林波¹, 王海燕¹, 刘香英¹, 张丹丹¹, 刘英嘉¹, 温得中²

1.吉林农业大学生命科学学院分子免疫学研究室|吉林长春 130118；2.吉林大学基础医学院医学遗传学教研室|吉林长春 130021

收稿日期:2009-01-21 出版日期:2009-07-28 发布日期:2009-08-24
通讯作者: 张林波 E-mail:cczlb@126.com
作者简介:张林波（1973-）|男|吉林省四平市人|医学博士|副教授|主要从事免疫学研究。
基金资助:
吉林省科技厅科研基金资助课题（20030224-2）

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

ZHANG Lin-Bo¹, WANG Hai-Yan¹, LIU Xiang-Ying¹, ZHANG Dan-Dan¹, LIU Ying-Jia¹, WEN De-Zhong²

1.Department of Immunology|College of Life of Sciences|Jilin Agricultural University,Changchun 130118|China；2.Department of Genetics,School of Basic Medical Sciences,Jilin University,Changchun 130021,China

Received:2009-01-21 Online:2009-07-28 Published:2009-08-24

摘要/Abstract

摘要：

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。

关键词: 结核分枝杆菌；随机肽库；模拟表位；免疫分析

Abstract:

Abstract: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 mixture of the positive clones as test group,BCG group as a positive control,TBS group as negative control group;Three weeks later,ELISA was used to detect the anti-PPD antibody titer in the immunized mice sera. The lymphocytes were separated from the immunized mice and the stimulating index (SI) was determined to further evaluate protective immune response to MTB.All the immunized mice were challenged with 10⁶ CFU BCG to study the protection conferred by the positive clones.Results After 3 rounds of effective bio-panning,15 of 20 phage clones were identified as positive clones by competent ELISA which could bind to polyclonal antibody IgG,the positive rate was 75%. A significant difference in valence of antibody in test group was observed compared with BCG group(P<0.01).In mouse spleen lymphocyte proliferation test,after stimulated by ConA,LPS,PPD,the SI values in test group were higher than those in BCG group,but there was no significant difference(P>0.05);the SI values were significantly higher than those in TBS group(P<0.01). The pathological changes of mouse lung section were observed in test group and BCG group, the alveolar tissue appeared to be intact with significantly less lymphocyte infiltration.In TBS group the pulmonary parenchyma developed severe fibrosis .The lotus bacterium (log CFU·mg^-1) 　in test group（4.080±0.035）was lower than that in TBS group（4.360±0.110）(P<0.01),but was higher than that in BCG group（3.980±0.023）. Conclusion Mycobacterium tuberculosis mimic epitopes of positive clones have been successfully screened.They can induce mouse to produce specific immunity and protective effect.This result laid a further experiment foundation for development of new tuberculosis vaccine of Mycobacterium tuberculosis.

Key words: Mycobacterium tuberculosis;phage display peptide library;mimotope;immunoassay

中图分类号:

Q939.13

张林波, 王海燕, 刘香英, 张丹丹, 刘英嘉, 温得中. 结核分枝杆菌抗原模拟表位肽的筛选及免疫活性鉴定[J]. J4, 2009, 35(4): 664-668.

ZHANG Lin-Bo, WANG Hai-Yan, LIU Xiang-Ying, ZHANG Dan-Dan, LIU Ying-Jia, WEN De-Zhong. Screening of Mycobacterium tuberculosis mimotopes from　 phage display peptide library and identification of immune activity[J]. J4, 2009, 35(4): 664-668.