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中国生物工程杂志

China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (12): 1-8    DOI: 10.13523/j.cb.20191201
    
Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori
GUO Le1,3,WANG Shu-e3,HE Meng3,ZHANG Fan3,LIU Hong-peng3,**(),LIU Kun-mei2,3,**()
1 Provincial Key Laboratory of Clinical Pathogenic Microbiology, General Hospital of Ningxia Medical University, Yinchuan 750004, China
2 Breeding Base of State Key Laboratory for Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750021, China
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Abstract  

Objective: To evaluate antigenic structure of multivalent epitope vaccine CWAE against Helicobacter pylori (H. pylori) by bioinformatics softs, obtain the purified CWAE protein after prokaryotic expression, and further identify the immunological properties of CWAE.Methods: The antigenic structure of CWAE was analyzed by bioinformatics softs. The recombinant plasmid pET28a-CWAE was obtained by using a synthetical WAE gene to replace the UE gene in pET28a-CUE plasmid. Then, the recombinant plasmid pET28a-CWAE was transformed into E. coli BL21 (DE3). After induction by IPTG, the antigen protein CWAE was purified by Ni-NTP nickel ion affinity chromatography. The adjuvant activity of CTB components in CWAE was identified by GM1-ELISA. Finally, the ability of CWAE to induce antibodies and lymphocyte immune response against H. pylori was detected by ELISA and spleen lymphocyte proliferation test.Methods: The multivalent epitope vaccine CWAE had a scientific and reasonable structure. The CWAE gene in recombinant plasmid pET28a-CWAE was consistent with the design sequence. After induction with IPTG, CWAE protein mainly exists as inclusion body. The purified CWAE was about 93.2% after purification.Results: from GM1-ELISA confirmed that CTB components in CWAE had good mucosal adjuvant activity. ELISA results confirmed that CWAE could stimulate BALB/c mice to produce anti-H. pylori antibodies. Moreover, CWAE vaccine could stimulate lymphocyte responses against various pathogenic factors from H. pylori.Conclusion: H. pylori multivalent epitope vaccine CWAE has scientific and reasonable antigen structure, and CWAE with high purity can be obtained by prokaryotic expression. Furthermore, the CWAE vaccine can stimulate specific antibodies and lymphocyte response against H. pylori. Experimental evidences for the development of multivalent epitope vaccine against H. pylori will be provided.



Key wordsHelicobacter pylori      Multivalent epitope vaccine      Specific antibody     
Received: 27 May 2019      Published: 15 January 2020
ZTFLH:  Q93  
Corresponding Authors: Hong-peng LIU,Kun-mei LIU     E-mail: 70576980@qq.com;lkm198507@126.com
Cite this article:

GUO Le,WANG Shu-e,HE Meng,ZHANG Fan,LIU Hong-peng,LIU Kun-mei. Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori. China Biotechnology, 2019, 39(12): 1-8.

URL:

http://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191201     OR     http://manu60.magtech.com.cn/biotech/Y2019/V39/I12/1

Fig. 1 Antigenic structure of CWAE vaccine and analysis of the linkers by DNAstar (a) Antigenic structure of CWAE vaccine (b) Analysis of the linkers (DPRVPSS, KK, GS and GGG) The linker (DPRVPSS, KK, GS and GGG) is marked in gray (c) Antigenicity analysis of CWAE
Fig.2 Construction and identification of recombinant plasmid pET28a-CWAE (a) The construction of recombinant plasmid pET28a-CWAE (b) PCR amplification of gene WAE (c) The pET28a-CWAE plasmids were digested with Kpn I and Xho I M:DNA marker; 1: The double restriction map (d) The pET28a-CWAE plasmids were digested with Nco I and Xho I M:DNA marker; 1: The double restriction map (e) Identification of plasmid pET28a-CWAE by gene sequencing Linker (DPRVPSS) is marked by a black line. Besides, the UreA27-53 Th epitope is marked by a red line
Fig.3 Expression and purification of antigen protein CWAE (a) Expression and purification of antigen protein CWAE M:Protein marker; 1:CWAE expression after induction for 6h; 2:CWAE expression after induction for 5h; 3:CWAE expression after induction for 4h; 4:CWAE expression after induction for 3h; 5:CWAE expression after induction for 2h (b) Purification of antigen protein CWAE M:Protein marker; 1:The inclusion body of CWAE; 2:The peak of loading protein sample; 3: The impurity protein eluted by wash buffer; 4: The purified CWAE protein washed down by elution buffer
Fig.4 Adjuvant activity of CTB component in CWAEwas identified by GM1-ELISA A GM1-ELISA was performed to evaluate the adjuvanticity of CTB component in CWAE vaccine. The CTB protein was used as a positive control. ELISA plates were coated with 1μg/well of GM1 ganglioside or BSA. The proteins CWAE or CTB were diluted from 100μg/ml to 0.78μg/ml
Fig.5 IgG and IgA antibodies against H. pylori after CWAE immunization (a) IgG antibodies against H. pylori lysates by ELISA (b) IgA antibodies against H. pylori lysates by ELISA.
Fig.6 Proliferation of splenic lymphocytes after CWAE immunization Splenic lymphocytes were separated from H. pylori-infected BALB/c mice after CWAE immunization, and were incubated with CWAE, Urease, UreA, UreB or H. pylori lysates
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