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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。
Objective To express Mycobacterium tuberculosis PPE68 protein in Escherichia coli and identify and purify recombinant rPPE68 protein to immunize New Zealand white rabbits to prepare polyclonal anti-PPE68 antibody. Methods The recombinant prokaryotic expression plasmid pET32a (+) - PPE68 was transformed into E. coli BL21. IPTG induced a large amount of PPE68 protein. The expressed protein was identified and purified by affinity chromatography. Purified recombinant rPPE68 was used as immunogen and mixed with equal volumes of Freund’s incomplete adjuvant. New Zealand white rabbits were immunized subcutaneously with multiple injections and blood was collected on the 7th day after the third immunization. The antiserum specificity was detected by agglutination test and Western-blot, and the antiserum titer was measured by two-way immunodiffusion method. Results The target product expressed in Escherichia coli was analyzed by SDS-PAGE, and the specific band was observed at 57 000 relative molecular mass. The immunoblot analysis confirmed that the target protein was expressed in the serum of mice infected with Mycobacterium tuberculosis H37Rv strain reaction. Purified rPPE68 protein immunized New Zealand white rabbits, agglutination reaction and Western-blot confirmed the specificity of the antiserum, serum titer measured by two-way immunodiffusion 1:16. CONCLUSION: Mycobacterium tuberculosis PPE68 protein has been successfully expressed and a polyclonal antibody specific against rabbit polyclonal antibody against PPE68 has been prepared. This provides a new idea for the early diagnosis of tuberculosis.