结核分枝杆菌Hsp16.3单克隆抗体的制备及其特性鉴定  

Production and Identification of Monoclonal Antibodies Against Mycobacterium tuberculosis Heat Shock Protein 16.3

作　　者：张彩勤[1] 江鹰[1] 赵勇[1] 毛峰峰[1] 赵善民[1] 白冰[1] 师长宏[1] 

机构地区：[1]第四军医大学实验动物中心,西安710032

出　　处：《科学技术与工程》2012年第1期30-33,共4页Science Technology and Engineering

基　　金：国家自然科学基金(NO.30972767);陕西省自然科学基金(2010JM4012)资助

摘　　要：制备抗结核分枝杆菌Hsp16.3的单克隆抗体,并对其生物学特性进行鉴定。将含目的基因的表达载体pProEXHTb-Hsp16.3,通过E.coli DH5α诱导表达,获得含有6×His的Hsp16.3蛋白,采用Ni-NTA纯化试剂盒进行目的蛋白的纯化,并用透析方法进行蛋白复性。将复性的蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合,间接ELISA筛选阳性杂交瘤细胞株。将获得的Hsp16.3阳性杂交瘤细胞株分别利用间接ELISA法和Western blot方法进行效价、相对亲和力及特异性的测定。获得了三株Hsp16.3的单克隆抗体,分别命名为3H6F9、1D5E1和4H8G6,其效价分别为1:1×107、1:1×106和1:1×106,相对亲和力分别为0.000 1 mg/mL、0.001 mg/mL和0.001 mg/mL,并且无交叉反应性。所获得的结核分枝杆菌Hsp16.3的单克隆抗体效价高、特异性强,为进一步研究Hsp16.3在结核分枝杆菌潜伏感染中的作用提供了有效的工具。To product monoclonal antibody against Hsp16.3 of Mycobacterium tuberculosis,and identify its character.The recombinant pProEXHTb-Hsp16.3 plasmid was transferred into E.coli DH5α,and induced by IPTG.The Hsp16.3 protein with 6×His was purified by Ni-NTA purification system under denaturing condition.Following by renaturation of dialysis,Hsp16.3 was used as antigen to immunize the BALB/c mice and product monoclonal antibodies.The positive clone was identified through indirect ELISA.The mAb specificity was assayed by ELISA and Western blot.Three anti-Hsp16.3 mAbs,named 3H6F9,1D5E1,4H8G6 respectively,were obtained.The titer and relative affinity of the three mAbs were 1：1×10^7,1：1×10^6,1：1×10^6 and 0.000 1 mg/mL,0.001 mg/mL,0.001 mg/mL.It could specially bind to Hsp16.3.The anti-Hsp16.3 mAbs with high specificity and high affinity were successfuly obtained,which were the effective tools to research the role of Hsp16.3 in Mycobacterium tuberculosis latent infection.

关 键 词：结核分枝杆菌 HSP16.3 单克隆抗体 

分 类 号：R521[医药卫生—内科学]