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

doi:10.13523/j.cb.2007040

China Biotechnology

2020, Vol. 40

Issue (11): 21-27 DOI: 10.13523/j.cb.2007040

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

HE Meng¹,ZHANG Guo-lin²,LI Yan¹,HAN Xue-bo¹,LIU Hong-peng¹,LI Xin¹,QIAN Ling-ling¹,LIU Kun-mei^3**(

),GUO Le^1**(

)

1 Provincial Key Laboratory of Clinical Pathogenic Microbiology, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
2 Suzhou Pharmaceutical Testing and Research Center, Suzhou 215000, China
3 Breeding Base of State Key Laboratory for Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750021, China

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Abstract

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.

Key words： Helicobacter pylori Pathogenicity island CagL Specific antibody

Received: 24 July 2020 Published: 11 December 2020

ZTFLH:

Q819

Corresponding Authors: Kun-mei LIU,Le GUO E-mail: lkm198507@126.com;guoletian1982@163.com

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	Meng HE
	Guo-lin ZHANG
	Yan LI
	Xue-bo HAN
	Hong-peng LIU
	Xin LI
	Ling-ling QIAN
	Kun-mei LIU
	Le GUO

Cite this article:

HE Meng,ZHANG Guo-lin,LI Yan,HAN Xue-bo,LIU Hong-peng,LI Xin,QIAN Ling-ling,LIU Kun-mei,GUO Le. Soluble Expression of Recombinant Antigen CagL from Helicobacter pylori Pathogenicity Island and Preparation and Analysis of Anti-CagA Polyclonal Antibody. China Biotechnology, 2020, 40(11): 21-27.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2007040 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I11/21

Fig.1 rCagL antigen structure and its bioinformatics analysis (a) rCagL antigen structure (b) 3D stereo structure predicted by Phyre2 (c) rCagL antigenicity analysis

Fig.2 Construction and identification of pCzn1-rCagL (a) Construction process of pCzn1-rCagL (b) The pCzn1-rCagL plasmids digested with Nde I and Xba I M: DNA marker; 1:The pCzn1-rCagL plasmids; 2:The digested pCzn1-rCagL plasmids (c) pCzn1-rCagL identification by gene sequencing

Fig.3 Expression and purification of rCagL (a) Expression of antigen protein rCagL M:Protein marker;1:rCagL expression without induction;2:rCagL inclusion proteins after induction; 3:rCagL soluble protein after induction;4:The whole bacterial proteins (b) Purification of rCagL M:Protein marker;1:The soluble protein of rCagL;2:The non-target proteins eluted by wash buffer;3:The purified rCagL eluted by elution buffer

Fig.4 The immunoreactivity of rCagL by Western blot analysis (a) rCagL reaction with anti-His antibody M:Protein marker;1: rCagL can react with anti-His antibody (b) rCagL reaction with anti-H. pylori antibody 1:rCagL can react with anti-H. pylori antibody;2:rCagL can’t react with normal rabbit serum

Fig.5 Specificity of anti-CagL polyclonal antibody (a) Anti-CagL polyclonal antibody against rCagL by ELISA (b) Anti-CagL polyclonal antibody against H. pylori lysates by ELISA


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