幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析

Soluble Expression of Recombinant Antigen CagL from Helicobacter pylori Pathogenicity Island and Preparation and Analysis of Anti-CagA Polyclonal Antibody

目的:利用原核表达和蛋白质纯化技术获得高纯度的幽门螺杆菌致病岛CagL重组抗原(rCagL),利用其制备anti-CagL多克隆抗体,并分析抗体的特异性。方法:通过生物信息学软件分析rCagL的抗原结构;利用PCR长片段DNA合成技术合成不含有信号肽序列的幽门螺杆菌致病岛CagL基因,将其插入表达质粒p Czn1中,构建重组质粒pCzn1-rCagL。然后,将pCzn1-rCagL转入大肠杆菌Arctic Express中,经IPTG诱导表达后,通过Ni-IDA镍离子亲和层析纯化重组抗原rCagL,利用Western blot鉴定rCagL与His标签抗体和Anti-H.pylori抗体的免疫反应性;最后,通过rCagL辅以弗氏佐剂免疫BALB/c小鼠,制备anti-CagL多克隆抗血清,通过ELISA方法分析抗血清的特异性。结果:生物信息学软件表明重组抗原rCagL具有较好的抗原性质;重组质粒pCzn1-rCagL经双酶切和基因测序等技术鉴定,证实rCagL核苷酸序列与理论序列完全一致;基因工程菌株pCzn1-rCagL/Arctic Express在低温11℃条件经IPTG诱导表达。SDS-PAGE实验结果证实:rCagL可实现相对高效地可溶性蛋白表达,可溶性蛋白约占包涵体的62.07%。经Ni-IDA亲和层析柱纯化,可获得高纯度rCagL,纯度约为96.6%。Western blot结果证实:重组抗原rCagL可特异性与His标签抗体和Anti-H.pylori抗体结合。ELISA结果证实:经rCagL免疫小鼠制备的多克隆抗体anti-CagL可特异性识别rCagL和H.pylori裂解物,具有较高的抗体特异性。结论:重组抗原rCagL在低温条件下可实现可溶性表达,经纯化可获得高纯度抗原蛋白;rCagL具有较好的抗原性,制备的多克隆抗体具有较好的免疫特异性,为发展H.pylori相关诊断试剂奠定了实验基础。

Objective:After prokaryotic expression of recombinant antigen CagL(rCagL)from Helicobacter pylori pathogenic island,recombinant antigen rCagL with high purity was obtained by protein purification technology.Anti-CagL polyclonal antibody was prepared and its specificity was also analyzed.Methods:The antigen structure of rCagL was analyzed by bioinformatics software.CagL gene without signal peptide was synthetized by PAS(PCR-based accurate synthesis),and then inserted into expression plasmid pCzn1 to construct recombinant plasmid pCzn1-rCagL.After that,the pCzn1-rCagL plasmids were transferred into E.coli Arctic Express.After expression with IPTG induction,the rCagL protein was purified by Ni-IDA affinity chromatography.The immunoreactivity of rCagL reaction with His label antibodies and anti-H.pylori antibodies was identified by Western blot.Lastly,anti-CagL polyclonal antibodies were prepared by immunizing BALB/c mice with rCagL plus Freund’s adjuvant,and the specificity of anti-CagL polyclonal antibodies was also analyzed by ELISA.Results:Bioinformatics software showed that recombinant antigen rCagL has good antigenic properties.The recombinant plasmid p Czn1-rCagL was identified by double enzyme digestion and gene sequencing,and the nucleotide sequence of rCagL was completely consistent with the theoretical sequence.After induction with IPTG at low temperature of 11℃,the recombinant genetically engineered strains pCzn1-rCagL/Arctic Express can express soluble rCagL protein efficiently by SDS-PAGE analysis.And the soluble rCagL protein accounted for 62.07%of inclusion bodies.The purity of the protein was about 96.6%after purification by Ni-IDA affinity chromatography.Western blot results confirmed that recombinant antigen rCagL could bind specifically to His label antibodies and anti-H.pylori antibodies.ELISA results showed that the prepared antiCagL polyclonal antibodies could specifically recognize rCagL and H.pylori lysates,indicating anti-CagL polyclonal antibodies had high antibody specificity.Conclusion:The recombinant antigen rCagL can be expressed at low temperature,and the rCagL protein with high purity was obtained after purification.The recombinant antigen rCagL had good antigenicity,and anti-CagL polyclonal antibodies had good immune specificity,which laid a foundation for the development of H.pylori-related diagnostic reagents.