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Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways., (© 2020 The Author(s).)

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin, but its practical application is limited due to the complex purification procedure. In this study, the C2 domain of streptococcal protein G (SPG) with the binding affinity for chicken IgY was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein, and purified to a high purity by inverse transition cycling (ITC). Binding experiments showed that chicken IgY could bind to and eluted off the ELP-C2 fusion protein in pH-, temperature- and/or time-dependent manners. By using the ELP-C2 protein, a simple chicken IgY purification method was developed, and its purification performance was compared with that of ammonium sulfate precipitation and ethanol fractionation. Quantitative SDS-PAGE analysis showed that the ELP-C2 binding capture method provided a chicken IgY purity of 96.3% and a recovery of 64%, both of which were significantly higher than that of the two traditional methods The ELP-C2 binding capture method could be accomplished within 3h, in contrast to 30.3h for ammonium sulfate precipitation or 4.3h for ethanol fractionation. These data suggest that the ELP-C2 binding capture was a simple, efficient and cost-effective method for purification of chicken IgY., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In recent years, the Chinese mitten crab (Eriocheir sinensis), which is widely farmed in China, has been frequently affected by Hepatospora eriocheir. The gut response of mitten crabs to H. eriocheir infection is not yet known. The aim of this study was to evaluate the effects of H. eriocheir infection on the gut microbiota and proteome of E. sinensis. In this study, E. sinensis was divided into two groups based on whether it was infected with H. eriocheir using PCR and Calcofluor white staining. Calcofluor white staining revealed that H. eriocheir was massively aggregated in the gut. Proteomic results showed that 3959 proteins were identified. Under a 1.2-fold change threshold, 420 differentially expressed proteins (DEPs) were screened, including 292 upregulated DEPs and 128 downregulated DEPs. Differential protein expression related to apoptosis and energy metabolism increased, while protein expression related to immune and inflammatory responses and cell motility decreased. In addition, the microbiota results showed that alpha diversity tended to increase, and beta diversity indicated significant differences in gut microbiota between the two groups. Bacteroidetes, Proteobacteria, and Firmicutes were the dominant phyla in the E. sinensis gut. Compared with the control group, Bacteroidetes were significantly reduced in the infected group, while Firmicutes and Tenericutes were significantly increased. At the genus level, the abundance of Bacteroides , Flavobacterium , Shewanella and Aeromonas decreased significantly, while Candidatus Bacilloplasma , Dysgonomonas , Tyzzerella_3 and Lactovum increased significantly. Microbial function prediction found that most differentially abundant bacteria were related to metabolism. At the same time, correlation analysis between the proteome and gut microbiota found potential roles of differential bacteria and proteins involved in metabolism and immunity. These findings suggest that H. eriocheir infection can alter the gut microbiota composition and proteome of E. sinensis. • Hepatospora eriocheir was massively aggregated in the Eriocheir sinensis gut. • 420 differential proteins were to H. eriocheir infection by TMT proteomics analysis. • Bacteroidetes reduced, while Firmicutes and Tenericutes increased in gut after H. eriocheir infection. • The differential bacteria and protein in H. eriocheir infection were involved in metabolism and immunity. [ABSTRACT FROM AUTHOR]