结核分枝杆菌PPE68蛋白的表达、鉴定及抗血清的制备

Prokaryotic expression and the antiserum preparation of PPE68 in mycobacterium tuberculosis

目的在大肠杆菌中表达结核分枝杆菌PPE68蛋白,鉴定并纯化重组rPPE68蛋白后免疫新西兰大白兔,制备兔抗PPE68多克隆抗体。方法将已经过鉴定的重组原核表达质粒pET32a(+)-PPE68转化至大肠杆菌BL21中,IPTG诱导大量表达PPE68蛋白。对表达蛋白进行鉴定后利用亲和层析法进行纯化。以纯化后重组rPPE68为免疫抗原,与弗氏不完全佐剂等体积混合,采用皮下多点注射法免疫新西兰大白兔,于第3次免疫后的第7天采血。用凝集实验与Western-blot检测抗血清的特异性,并用双向免疫扩散法测定抗血清效价。结果在大肠杆菌中表达的目的产物经SDS-PAGE分析,在相对分子质量57 000处可见特异性条带,免疫印迹分析证实表达的目的蛋白可与结核分枝杆菌H37Rv株感染小鼠的血清发生反应。纯化的rPPE68蛋白免疫新西兰大白兔后,凝集反应与Western-blot证实了抗血清的特异性,双向免疫扩散法测得血清效价为1∶16。结论已成功表达结核分枝杆菌PPE68蛋白,制备了特异性兔抗PPE68多克隆抗体,对结核病的早期诊断提供了一种新的思路。

This study is aimed to construct the prokaryotic expression vector of PPE68 of Mycobacterium tuberculosis and purify the PPE68 protein to prepare antiserum against PPE68.After identifying by DNA sequencing,the constructed recombinant plasmid was transformed into E.coli BL21 and the recombinant protein was expressed after induction with IPTG.The target protein was identified by SDS-PAGE and Western-blot,and purified by affinity chromatograph.Rabbit antiserum against PPE68 was prepared by hypodermic immunization mixed with Freund＇ incomplete adjuvant and the titer was detected by agglutination test,Western-blot and double immunodiffusion.SDS-PAGE showed specific protein band with a relative molecular mass of 57 000.The protein can bind with the antibody in the blood serum of the mouse immuned by Mycobacterium tuberculosis.The specificity of the rabbit antiserum was determined by agglutination test and Western-blot.The antiserum had a double immunodiffusion titer about 1∶16.The recombinant protein PPE68 and the rabbit antiserum against it were prepared successfully,which may be applied in PPE68 further function research.