Immune enhancement of Taishan Robinia pseudoacacia polysaccharide on recombinant Proteus mirabilis OmpA in chickens

https://doi.org/10.1016/j.intimp.2014.06.036Get rights and content

Highlights

  • We firstly expressed Proteus mirabilis OmpA in cells of the Pichia pastoris.

  • Evaluated the effects of TRPPS on chickens immunized with recombinant OmpA vaccine.

  • Established that the optimal dose of TRPPS was 200 mg/mL for immune responses.

Abstract

This study was conducted to evaluate the effects of Taishan Robinia pseudoacacia polysaccharide (TRPPS) on immune responses of chickens immunized with Proteus mirabilis outer membrane protein A (OmpA) recombinant protein vaccine. OmpA was expressed in Pichia pastoris and mixed with TRPPS. 360 chickens were randomly divided into six groups. Groups I to IV were treated with OmpA which contained TRPPS of three different dosages, Freund's adjuvant, respectively. Groups V and VI were treated with pure OmpA and physiological saline, respectively. The data showed that the antibody titers against OmpA, the concentration of IL-2, CD4 +, and CD8 +, T lymphocyte proliferation rate in Group II were significantly higher (P < 0.05) than those in the other groups, little difference in SIgA content was observed among groups I to VI. These results indicated that TRPPS strengthened humoral and cellular immune responses against recombinant OmpA vaccine. Moreover, 200 mg/mL TRPPS showed significance (P < 0.05) compared with Freund's adjuvant. Therefore, TRPPS can be developed into an adjuvant for recombinant subunit vaccine.

Introduction

Proteus mirabilis (P. mirabilis) is a well-known Gram-negative opportunistic pathogen [1]. This bacterium can infect various day-old chicks and cause many clinical symptoms such as poultry diarrhea, paralysis of the limbs, sepsis, and encephalomalacia. The pathogen spreads by horizontal or vertical transmission and can easily lead to outbreaks of epidemic diseases [2]. Presently, few effective vaccines were available to protect chickens against P. mirabilis. Previous studies had proven that the outer membrane protein A (OmpA) of P. mirabilis shows good immunogenicity [3]. Hence, a recombinant subunit vaccine could be developed for P. mirabilis.

Adjuvants are nonspecific immuno-enhancers that are used to strengthen the immune response of the body or modulate immune response types [4]. Existing adjuvants can enhance the immunogenicity of highly purified or recombinant antigens, but they pose a high risk of adverse side effects and are expensive [5]. Plant polysaccharide has gained much attention because of its diversity, abundance, and effectiveness [6]. A number of plant polysaccharides, such as Epimedium, Lycium barbarum, and Enteromorpha prolifera polysaccharide, possess immune-enhancement effects and are excellent candidates to replace common adjuvants for many vaccines [7], [8], [9]. Robinia pseudoacacia is a traditional Chinese medicine material that has been used orally since the Song Dynasty. This material has high pharmaceutical value because of its antibacterial and anti-inflammatory activities, thereby promoting health [10]. We have previously demonstrated that Taishan R. pseudoacacia polysaccharide (TRPPS) can improve the development of the immune system, as well as increase the indices of immune organs and serum antibody titers against NDV, in chicks [11]. However, there are no researches on the immune effects of TRPPS to recombinant protein vaccine.

In present research, we produce a recombinant protein of P. mirabilis OmpA in Pichia pastoris (P. pastoris). TRPPS was used as the adjuvant for recombinant protein vaccine. The purpose of this research is to evaluate the effects of TRPPS on immune responses of chickens immunized with P. mirabilis OmpA recombinant protein vaccine.

Section snippets

Strains, plasmids, and media

P. mirabilis strain Q1 from chicks was the bacterial strain used in this study. This strain was identified and preserved previously by the Animal Biotechnology and Disease Control and Prevention Laboratory of Shandong Agriculture University. Escherichia coli DH5α, P. pastoris and the plasmid pPIC9 were provided by Invitrogen (Carlsbad, CA, USA). The media used in this study were prepared according to the Pichia expression manuals [12].

Expression, purification, and identification of P. mirabilis OmpA

To amplify OmpA genes, a pair of primers (P1:

P. mirabilis OmpA expression in P. pastoris

The results revealed that the OmpA gene was correctly cloned into the expression vector pPIC9 and then transformed into P. pastoris. A novel protein band corresponding to 52 kDa, which was identical with the predicted result, was detected in P. pastoris/pPIC9-OmpA transformants by SDS-PAGE (Fig. 1a). The protein first appeared in the 48 h cell culture supernatant. The productivity increased and reached 8 μg/mL in the 96 h cell culture supernatant. A similar molecular weight of approximately 52 kDa

Discussion

P. pastoris expression system has been extensively used in recombinant protein research. This system is characterized by easy cultivation, high levels of productivity, and the property of performing eukaryotic post-translational modifications [20]. The efficiency of yeast-expressed antigen has been investigated in several animals for the development of bacterial vaccines [21], [22]. The expressed OmpA of P. mirabilis could remove many of the components irrelevant to immunity, thereby displaying

Conclusion

Our findings demonstrated that TRPPS could enhance the effects of the recombinant subunit vaccine, such as by stimulating the body to develop enough immunity against pathogenic microorganisms and inducing better humoral and cellular immunities compared with the commonly used Freund's adjuvant. We further established that the optimal dose of TRPPS was 200 mg/mL. TRPPS may have protective effects on fighting P. mirabilis by increasing antibody titers against OmpA. Moreover, TRPPS could be used as

Acknowledgments

The project was supported by the National Science Foundation of China (Foundation No. 31272595), the Science and Technology Development Plan of Shandong Province (2012GNC11020), and the Science and Technology Develop Project in Taian (20103001). We are grateful to all the staff in the Institute of Animal Science and Technology of Shandong Agricultural University for their assistances in the study.

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