Construction and evaluation of a novel triple cell epitopebased polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and Streptococcus

Xiang Liu,Chunlin Chen,Gregory Marslin,Chen Chen,Rui Ding,Sanqiao Wu

doi:10.4314/tjpr.v16i10.23

Abstract

Purpose: To construct a novel triple cell epitope-based polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and Streptococcus and to reduce the use of antibiotics.Methods: Based on bioinformatics approach, a novel triple epitope-based polypeptide (CM-TEP) was designed and subjected to Ni-NTA flow resin purification. Purified CM-TEP was immunized into mice to prepare a polyclonal antibody. Pull-down assays and enzyme-linked immunosorbent assay (ELISA) were used to detect the interaction between CM-TEP antibodies and S. aureus, E. coli and Streptococcus. Active immunity mice and challenge of bacterial pathogens were used to detect immune protection of CM-TEP. Additionally, the optimal expressing conditions of CM-TEP strain were analyzed using orthogonal test design.Results: A novel cow mastitis triple cell epitope-based polypeptide (CM-TEP) with a MW of 36 kDa was designed, purified and used to immunize mice to prepare a polyclonal antibody. Pull-down assays and ELISA data showed that CM-TEP antibodies directly interacted with S. aureus, E. coli and Streptococcus. CM-TEP displayed a significant immune protective effect against infection by S. aureus (50 %, p < 0.05) and E. coli (54.54 %, p < 0.05) and provided some immune protective effect (30.78 %, p > 0.05) against Streptococcus. The optimum expressing conditions of CM-TEP were as follows: IPTG concentration of 0.3 mmol/L, strain OD600 value of 1, inducing temperature of 37 oC, and inducing time of 8 h.Conclusion: The findings suggest that epitope-based vaccine of CM-TEP may be a useful strategy for treating cow mastitis induced by S. aureus, E. coli and Streptococcus.Keywords: Cow mastitis, Epitope vaccine, Immunogenicity, Immune protective

Highlights

Cow mastitis is the most frequent and costly disease affecting dairy products worldwide [1], and its main pathogens include Staphylococcus aureus (S. aureus), Streptococcus and Escherichia coli (E. coli)
On the basis of the common epitope sequence predicted by the two programs, the B cell epitope segment of S. aureus clumping factor A (ClfA) protein was at residues 114 – 129 and 326 – 330, and that of the Ebps protein was at residues 87 – 102 and 132 – 147
The B cell epitope segment of E. coli outer membrane protein A (OmpA) protein was at residues 44 – 50 and 126 – 135, and that of the outer membrane protein C (OmpC) protein was at residues 19 – 31 and 129 – 144

Summary

INTRODUCTION

Cow mastitis is the most frequent and costly disease affecting dairy products worldwide [1], and its main pathogens include Staphylococcus aureus (S. aureus), Streptococcus and Escherichia coli (E. coli). This study sought to construct and evaluate a novel triple-cell epitope-based vaccine for cow mastitis (CM-TEP), using these six proteins. CM-TEP nucleic acid sequences were synthesized and cloned in E. coli BL21 by the Shanghai Xuguan Biotech Development Corp (Shanghai, China). The optimal antigenic combination was chosen as the final triple epitope polypeptide vaccine for cow mastitis (CM-TEP). CM-TEP nucleic acid sequences were synthesized and cloned in E. coli BL21 by the Shanghai Xuguan Biotech Development Corp., China. Five-week-old Kunming mice were administered purified protein (100 μg per mouse) emulsified with Freund’s Complete Adjuvant Mice were randomly divided into groups, and purified CM-TEP protein was intraperitoneally injected two times at an interval of 10 days, to deliver the primary and booster doses.

RESULTS

DISCUSSION

CONCLUSION

Conflict of Interest