解脲支原体1型MB抗原保守区基因片段的克隆、表达及纯化

Expression and Purification of the Conserved Segment of MB Antigen Gene of Ureaplasma Urealyticum, Serotype - 1 in E. coli

目的 以解脲支原体1型培养液为模板，PER获得其MB抗原保守区部分基因，进行体外表达和纯化。方法用ExPasy网上软件Protscale分析Uu14个血清型的MB蛋白结构特点及抗原特性，选定14个血清型均在该蛋白同一保守区域有较好的抗原性，针对该序列设计特异性引物，并以UU血清1型DNA为模板进行PER，获得其MB抗原基因相应的片断，克隆入表达载体经测序鉴定后。在大肠杆菌中表达并纯化，以Western—blot鉴定表达产物。结果 准确扩增了解脲支原体1型MB抗原保守区基因片段，并在大肠杆菌中获得表达。结论 成功构建了pQE30／MB抗原部分保守序列的原核表达载体，在体外成功表达并纯化。表达和纯化的产物具有免疫反应性。

Objective To acquire the conserved segment of multiple - banded （MB） antigen gene of ureaplasma urealyticum,serotype 1 by PCR, expressed its DNA in E. coli and purified the expressed products. Methods Using Protscale software in the ExPasy, analyzed the MB antigens of all serotypes of ureaplasma urealytieum and ehosed part of conserved segment which antigenic epitope gatherd region. Using specific primers to amplify the target DNA segment as DNA template from culture Uu serotype 1 by PCR. After cloning into vector of pUCm - T ,it was sequenced. Then it was subeloned into expression vector pQE30 and expressed in E.coli by IPTG induce. Used Ni- NTA Spin kit in different conditions to purify expression products which existed in the supernate as soluble form. The expression and purified products were identified by SDS- PAGE and Western blot. Results The target DNA of the conserved segment of MB antigen was acquired by PCR. The gene segment could be expressed in E. coll. Conclusion Prokaryofie expression vector pQE30/partMBA is constructed, purely expressed products were obtained and the expressed product have the immune reactivity.