抗结核分枝杆菌抗原优势肽段单克隆抗体的制备和初步鉴定

Development and Characterization of Monoclonal Antibodies against the fused dominant peptide antigen of Mycobacterium tuberculosis

目的:制备和初步鉴定抗结核分枝杆菌抗原优势肽段的单克隆抗体。方法:以两种优势肽段的融合抗原PGEX-4T-2/38kD-ESAT6-CFP10和PGEX-4T-2/Mtb8.4-MPT64-Mtb16.3-Mtb8作为免疫原免疫BALB/c小鼠,取其脾细胞与SP2/0小鼠骨髓瘤细胞融合后,通过有限稀释法筛选阳性克隆,经ELISA和Western blotting分析单抗的特性和特异性。结果:获得17株结核分枝杆菌抗原优势肽段特异单克隆抗体杂交瘤细胞株,经间接ELISA方法测定,杂交瘤细胞培养上清效价为28～212,接种小鼠的腹水效价为216～220。Western blotting结果显示,每株单抗均能特异性的识别融合蛋白的优势肽段。结论:制备了针对抗结核分枝杆菌抗原优势肽段的单克隆抗体,为结核分枝杆菌的快速诊断和抗原性分析奠定了基础。

Objective：To develop and characterize monoclonal antibodies（McAbs） against the dominant peptide antigen of Mycobacterium tuberculosis.Methods： BALB/C mice were immunized with two fused dominant peptide antigen（PGEX-4T-2/38kD-ESAT6-CFP10,PGEX-4T-2/ Mtb8.4-MPT64-Mtb16.3-Mtb8） of Mycobacterium tuberculosis.Splenocytes from the immunized mice were fused with SP2/0 myeloma cells.Hybridoma culture supernatants were screened and evaluated by ELISA.Positive clones were checked for isotype and McAbs were confirmed by Western blotting analysis.Results： Seventeen strains of stable hybridoma cells secreting Mcab against the fused dominant peptide antigen of Mycobacterium tuberculosis were obtained.The titers of these Mcabs in the culture supernatants were 28-212,and the tietrs in the ascites of the mice were 216-220 detected by ELISA.The results of western blotting showed that each Mcab only reacted with the dominant peptide of the fused antigen.Conclusion： McAbs specific to fused dominant peptide antigen of Mycobacterium tuberculosis were developed.McAbs would be helpful for rapid clinical diagnosis and antigenic epitope analysis for Mycobacterium tuberculosis.