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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (4): 34-41    DOI: 10.13523/j.cb.1910009
技术与方法     
以核糖体蛋白L7/L12为分子标志物精准检测肺炎链球菌的研究 *
王猛1,宋慧茹1,程雨洁1,王毅1,2,3,杨波1,2,3,胡征1,2,3,**()
1 湖北工业大学生物工程与食品学院 武汉 430068
2 发酵工程教育部重点实验室(湖北工业大学) 武汉 430068
3 工业发酵湖北省协同创新中心 武汉 430068
Accurate Detection of Streptococcus pneumoniae by Using Ribosomal Protein L7 / L12 as Molecular Marker
WANG Meng1,SONG Hui-ru1,CHENG Yu-jie1,WANG Yi1,2,3,YANG Bo1,2,3,HU Zheng1,2,3,**()
1 School of Food and Biological Engineering, Hubei University of Technology, Wuhan 430068, China
2 Key Laboratory of Fermentation Engineering (Hubei University of Technology), Ministry of Education, Wuhan 430068, China
3 Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430068, China
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摘要：

目的：以核糖体蛋白L7/L12为标志物,探索一种胶体金免疫层析法快速检测肺炎链球菌的方法。方法：分析核糖体蛋白L7/L12基因序列,构建原核表达载体,表达纯化重组蛋白,免疫BALB/c小鼠制备单克隆抗体;利用无标记分子相互作用仪检测抗原抗体亲和力;以双抗夹心法研制胶体金免疫层析检测试纸条,并对其特异性、敏感性及稳定性进行验证分析。结果：筛选得到两株高效分泌单克隆抗体的杂交瘤细胞株,两种单克隆抗体均与抗原有高的亲和力并且无竞争,制作的试纸最低检出限为1.0×105 CFU/ml;其与肺炎链球菌有特异性反应,而与其它9种常见呼吸道病原菌均无交叉反应;试纸条在25℃下保存12个月仍具有良好的重复性和稳定性。结论：RP-L7/L12可作为肺炎链球菌的检测标志物,以其单抗制备的胶体金免疫层析试纸可适用于肺炎链球菌的快速检测。
关键词： 肺炎链球菌;  核糖体蛋白L7/L12;  单克隆抗体;  胶体金免疫层析法    
Abstract:

Objective: A rapid colloidal gold immunochromatography assay (GICA) for the detection of Streptococcus pneumoniae was explored by using the ribosomal protein L7/L12 as detection marker. Method: The gene sequence of ribosomal protein L7/L12 was analyzed, the prokaryotic expression vector was constructed, the recombinant protein was expressed and purified, and the monoclonal antibody was prepared by immunizing BALB/c mice; The affinity of antigens and antibodies was detected by a labelless molecular interaction instrument based on BLI technology; The colloidal gold immunochromatographic test strip was developed based on the principle of double antibody sandwich, and its specificity, sensitivity and stability were evaluated. Result: Two hybridoma cell strains that can efficiently secrete anti-monoclonal antibody were acquired by means of screening. Purified monoclonal antibodies all had high affinity and no competition with antigen. Pairing and detection were made on colloidal gold immunochromatography platform. The minimum detection limit of the test strips was 1.0×105 CFU/ml; It had a specific reaction with Streptococcus pneumoniae, but did not cross-react with other 9 kinds of common respiratory pathogens such as Haemophilus influenzae and Moraxella catarrhalis; Test strips were kept at 25°C for 12 months and still had good repeatability and stability. Conclusions: RP-L7/L12 can be used as a detection marker for Streptococcus pneumonia. The colloidal gold immunochromatographic test strips prepared by its monoclonal antibody can be used for rapid detection of streptococcus pneumoniae.
Key words: Streptococcus pneumoniae    Ribosomal protein L7/L12    Monoclonal antibody    Colloidal gold immunochromatography
收稿日期: 2019-10-10 出版日期: 2020-05-18
ZTFLH:  Q819  
基金资助: * 湖北省科技厅自然科学青年基金项目(2017CFB226)
通讯作者: 胡征     E-mail: zhenghu@mail.hbut.edu.cn
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王猛
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引用本文:

王猛,宋慧茹,程雨洁,王毅,杨波,胡征. 以核糖体蛋白L7/L12为分子标志物精准检测肺炎链球菌的研究 *[J]. 中国生物工程杂志, 2020, 40(4): 34-41.

WANG Meng,SONG Hui-ru,CHENG Yu-jie,WANG Yi,YANG Bo,HU Zheng. Accurate Detection of Streptococcus pneumoniae by Using Ribosomal Protein L7 / L12 as Molecular Marker. China Biotechnology, 2020, 40(4): 34-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1910009    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I4/34

Primer sequences Restriction enzyme cutting site
F 5'-TTAGGATCCATGGCATTGAACATTGAAAA-3' BamHI
R 5'-ATACTCGAGTTTAAGAGTAACTGAAGCTC-3' XhoI
表1  PCR扩增的引物序列
图1  Sp-RP-L7/L12的诱导表达及纯化结果
图2  抗体纯化SDS-PAGE鉴定分析
图3  间接ELISA法测定抗体效价
Subtype Lp-1 Lp-2
H-chain IgG1 1.043 2 1.429
IgG2a 0.249 9 0.214 5
IgG2b 0.145 2 0.204 8
IgG3 0.068 2 0.063 2
IgA 0.13 0.088 4
IgM 0.097 4 0.091 5
L-chain Kappa 0.500 3 0.616
Lambda 0.086 4 0.064 7
表2  单克隆抗体亚型鉴定
图4  Western blotting鉴定单抗SP-1#和SP-2#
图5  分子相互作用仪检测抗体亲和力
Conc. (nM) Response KD (M) kon(1/Ms) kdis(1/s) Full R2
SP-1# 1 000 4.876 1 9.05E-09 2.64E+04 2.39E-04 0.997 1
500 3.897 7 8.86E-09 3.25E+04 2.88E-04 0.997 9
250 2.819 6 8.12E-09 4.38E+04 3.56E-04 0.998 3
125 2.442 8 9.29E-09 4.68E+04 4.35E-04 0.993 2
62.5 1.546 5 5.91E-09 6.92E+04 4.09E-04 0.998 1
SP-2# 1 000 3.887 7 3.67E-09 5.96E+04 2.19E-04 0.995 1
500 2.806 0 5.36E-09 7.89E+04 4.23E-04 0.992 8
250 1.900 2 6.51E-09 8.89E+04 5.79E-04 0.992 5
125 1.410 1 7.72E-09 4.05E+04 3.13E-04 0.993 1
62.5 0.698 3 4.57E-09 9.36E+04 4.28E-04 0.991 2
表3  分子相互作用仪检测数据结果
图6  随机法验证Sp抗体识别不同表位
图7  肺炎链球菌胶体金免疫层析试纸条的特异性检测
图8  肺炎链球菌胶体金免疫层析试纸条的灵敏性检测
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