结核分枝杆菌Hsp16.3单克隆抗体的制备及其特性鉴定

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譎is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?07、1:1?06和1:1?06，相对亲和力分别为0.0001 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。

Production and identification of monoclonal antibodies against Mycobacterium tuberculosis heat shock protein 16.3

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 1:1×107,1:1×106,1:1×106 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.