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*

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pCzn1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。

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

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 pCzn1-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 anti-CagL 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.