重组幽门螺旋杆菌黏附素A的可溶性表达、纯化及其免疫学性质分析

Soluble expression,purification and immunological properties of recombinant H.pylori adhesin A

目的 通过分子克隆、蛋白表达及纯化等技术获得重组幽门螺旋杆菌黏附素A(recombinant H.pylori adhesin A,rHpaA),免疫BALB/c小鼠,制备anti-HpaA多克隆抗体后,分析其抗体特异性。方法 利用Phyre2和DNAstar生物信息学软件分析rHpaA的三维结构及抗原性质;通过PCR长片段DNA合成技术获得黏附素HpaA基因,插入质粒pCzn1中,制备重组质粒pCzn1-rHpaA,转化E.coli Arctic Express(DE3),经IPTG诱导表达、Ni-IDA亲和层析纯化后,获得rHpaA蛋白,Western blot鉴定其反应原性。将rHpaA辅以弗氏佐剂免疫雄性BALB/c小鼠,共6只,制备antiHpaA多克隆抗体,ELISA法鉴定其抗体特异性。结果 rHpaA具有较好的三维结构及抗原性质。双酶切鉴定及基因测序证明,重组质粒pCzn1-rHpaA含有完全正确的HpaA基因序列。重组菌株pCzn1-rHpaA/Arctic Express可溶性表达目的蛋白rHpaA,约占上清总蛋白的68.3%,纯度达98.1%。rHpaA可与anti-His和anti-H.pylori抗体相结合;antiHpaA多克隆抗体可特异性识别rHpaA和H.pylori裂解物。结论 经低温诱导表达和蛋白纯化等技术,可获得高纯度目的蛋白rHpaA,制备的anti-HpaA多克隆抗体具有良好的特异性,为研究H.pylori相关诊断试剂奠定了实验基础。

ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.