Dataset Information

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants.

ABSTRACT: Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (?1 and ?2) with one of the two LC types (k and ?) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HC?1 and LC? provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 ?g IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.

SUBMITTER: Juarez P

PROVIDER: S-EPMC3634489 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants.

Juarez Paloma P Huet-Trujillo Estefania E Sarrion-Perdigones Alejandro A Falconi Erica Elvira EE Granell Antonio A Orzaez Diego D

International journal of molecular sciences 20130318 3

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen ...[more]

PMID: 23507755

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Project description:We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated strain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mIL4/47) on either side of the inserted coding sequence and, in the other case, nonidentical sequences that varied at the nucleotide level without changing the amino acid sequences (CVB3-PL2-mIL4/46). Transfection of HeLa cells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were approximately 10-fold lower than those of the parental virus. Western blot analysis of viral proteins isolated from HeLa cells inoculated with either strain of chimeric virus demonstrated that the chimeric viruses synthesized capsid protein 1D at approximately twofold-higher levels than the parental virus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELISA) in HeLa cells inoculated with either strain of chimeric virus. Lysates of HeLa cells inoculated with either chimeric virus induced the proliferation of the mIL-4-requiring murine MC-9 cell line, demonstrating biological activity of the CVB3-expressed mIL-4. Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa cells demonstrated deletion of the mIL-4 coding sequence occurring by the fourth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated detection of the mIL-4 coding sequence in the virus population through 10 generations in HeLa cells. mIL-4 protein levels determined by ELISA were consistent with the stability and loss data determined by RT-PCR analysis of the passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 during replication in mice showed the presence of the viral mIL-4 insert in pancreas, heart, and liver at 14 days postinfection. Comparison of the murine antibody responses to CVB3-PL2-mIL4/46 and the parental CVB3/0 strain demonstrated an increased level of CVB3-binding serum immunoglobulin G1 in mice inoculated with CVB3-PL2-mIL4/46.

Project description:Background:With the continuous improvement of the modernization of the Chinese military and the major adjustments made by the state to the recruitment policy, the newly recruited military undergone multiple pressures such as targeted high-intensity military training and environmental changes. The mental health of military has become a crucial factor of improving the fighting capacity effectiveness of the troops. Objectives:To explore occupational stress of young recruits in the Xinjiang plateau environment during their basic military training period and analyze the relationship between occupational stress and secretory immunoglobulin A (sIgA) levels. Methods:Using multistage stratified cluster random sampling, 625 recruits stationed at Xinjiang plateau command in 2014 were enrolled as subjects. Occupational stress was assessed by the Occupational Stress Inventory Revised Edition (OSI-R). sIgA in saliva was quantified by enzyme-linked immunosorbent assay. The resulting data were analyzed using descriptive statistics, nonparametric tests, and correlation analysis. Results:Based on demographic characteristics, occupational stress was higher in the urban group than the rural group, coping ability for stress was greater in individuals who were students before joining the army than nonstudents, occupational stress was higher in smokers than nonsmokers, and coping ability for stress was higher in nonsmokers than in smokers (all P < 0.05). Being an only child, educational level and age were not significantly related to occupational stress scores (P > 0.05). Salivary sIgA level was higher in the high occupational stress group than in the low stress group (P < 0.01). Salivary sIgA was positively correlated with scores on the occupational role and personal strain questionnaires (r s = 0.229, r s = 0.268, P < 0.01). Conclusion:Demographic characteristics influenced occupational stress among young recruits in cold and high-altitude area. Further, there were some relationships between occupational stress and salivary sIgA in young military recruits.

Dataset Information

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants.

Publications

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants.

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