Protective Immunity of Pichia pastoris-Expressed Recombinant Envelope Protein of Japanese Encephalitis Virus

Abstract

Japanese encephalitis virus (JEV) envelope (E) protein holds great promise for use in the development of a recombinant vaccine. Purified recombinant E (rE) protein may be useful for numerous clinical applications; however, there are limitations in using the Escherichia coli expression system for producing high-quality rE protein. Therefore, in this study, the yeast expression system was used to generate the rE protein. For protein production using the yeast system, the full-length JEV E gene was cloned into Pichia pastoris. SDS-PAGE and immunoblotting analysis demonstrated that the rE protein had a molecular mass of 58 kDa and was glycosylated. The predicted size of the mature unmodified E protein is 53 kDa, suggesting that post-translational modifications resulted in the higher molecular mass. The rE protein was purified to greater than 95% purity using combined ammonium sulfate precipitation and a SP-Sepharose Fast Flow column. This purified rE protein was evaluated for immunogenicity and protective efficacy in mice. The survival rates of mice immunized with the rE protein were significantly increased over that of Hyphantria cunea nuclear polyhedrosis virus E protein (HcE). Our results indicate that the rE protein expressed in the P. pastoris expression system holds great promise for use in the development of a subunit vaccine against JEV.

Recent advances in the production of recombinant subunit vaccines inPichia pastoris

“…Japanese encephalitis virus (JEV) envelope (E) protein expressed in P. pastoris was glycosylated. 71 The purity of E protein was greater than 95%. P. pastorisproduced recombinant E protein could stimulate immune responses that were capable of mediating significant protection against JEV infection.…”

Secretory Expression Of The Glucan Endo-1,3-Beta-D-Glucosidase Gene Of Secale Cereale In Yeast Pichia Pastoris

“…Ammonium sulfate precipitation is often the first step in the purification of untagged recombinant proteins from yeast culture [37]. This approach allows to significantly concentrate the recombinant protein and get rid of some impurity proteins, while not disrupting the structure and properties of the target proteins [38,39]. It was experimentally found that recombinant ScGlu-3 is precipitated only when the concentration of ammonium sulfate reaches 50-65% (Fig.…”

Avaliação De Métodos Para Obtenção De Proteínas Recombinantes

“…Neste sentido, cabe salientar que a reação de anticorpos bovinos específicos evidenciou uma maior reatividade à rBm86-CG obtida com sulfato de amônio em comparação a acetona e metanol. Além disso, a precipitação por sulfato de amônio permite que a amostra seja concentrada e submetida à purificação por cromatografia sem prejuízos a sua imunogenicidade (JEONG et al, 2016;KWON, 2012;LI et al, 2015;MA, 2013).…”