Discovery of Missing Proteins from an Aneuploidy Cell Line Using a Proteogenomic Approach.

With the steadfast development of proteomic technology, the number of missing proteins (MPs) has been continuously shrinking, with approximately 1470 MPs that have not been explored yet. Due to this phenomenon, the discovery of MPs has been increasingly more difficult and elusive. In order to face this challenge, we have hypothesized that a stable aneuploid cell line with increased chromosomes serves as a useful material for assisting MP exploration. Ker-CT cell line with trisomy at chromosome 5 and 20 was selected for this purpose. With a combination strategy of RNA-Seq and LC-MS/MS, a total of 22 178 transcripts and 8846 proteins were identified in Ker-CT. Although the transcripts corresponding to 15 and 15 MP genes located at chromosome 5 and 20 were detected, none of the MPs were found in Ker-CT. Surprisingly, 3 MPs containing at least two unique non-nest peptides of length ≥9 amino acids were identified in Ker-CT, whose genes are located on chromosome 3 and 10, respectively. Furthermore, the 3 MPs were verified using the method of parallel reaction monitoring (PRM). These results suggest that the abnormal status of chromosomes may not only impact the expression of the corresponding genes in trisomy chromosomes, but also influence that of other chromosomes, which benefits MP discovery. The data obtained in this study are available via ProteomeXchange (PXD028647) and PeptideAtlas (PASS01700), respectively.

[Preparation of pan-neutralizing mouse monoclonal antibodies against enterovirus 71 and coxsackievirus A16].

Objective To prepare a neutralizing monoclonal antibody (mAb) that can simultaneously block enterovirus 71 (EV71) and coxsackievirus A16 (CV-A16) infections. Methods BALB/c mice were immunized with 163-177 amino acids (SP55) of the C-terminal of EV71 virion particle 1 (VP1) protein, and the mAbs were prepared by hybridoma technology. Neutralization antigenic epitope SP55 of EV71 and the highly homologous CV-A16 VP1 protein C-terminal 163-177 amino acids (PEP55) were applied to detect the mAbs that cross-reacted with EV71 and CV-A16 at the same time, and an in vitro neutralization test was conducted to detect the neutralization effect of EV71 and CV-A16, and to analyze the biological characteristics of the mAb. Results A mAb 6E5 with IgG1 subclass heavy chain and Kappa light chain was prepared, 6E5 mAb can cross-neutralize both EV71 and CV-A16. The mAb 6E5 could neutralize EV71 with a titer of 1:128, and CV-A16 with a titer of 1:32. Conclusion We have prepared a mAb 6E5 with a pan-spectrum neutralizing activity that can neutralize EV71 and CV-A16 at the same time.

A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs.

Coxsackievirus A6 (CVA6) is recognized as a major enterovirus type that can cause severe hand, foot, and mouth disease and spread widely among children. Vaccines and antiviral drugs may be developed more effectively based on a stable and easy-to-operate CVA6 mouse infection model. In this study, a wild CVA6-W strain was sub-cultured in newborn mice of different ages (in days), for adaptation. Therefore, a CVA6-A mouse-adapted strain capable of stably infecting the mice was generated, and a fatal model was built. As the result indicated, CVA6-A could infect the 10-day-old mice to generate higher levels of IFN-γ, IL-6, and IL-10. The mice infected with CVA6-A were treated with IFN-α1b at a higher dose, with complete protection. Based on this strain, an animal model with active immunization was built to evaluate antiviral protection by active immunization. The three-day-old mice were pre-immunized with inactivated CVA6 thereby generating IgM and IgG antibodies within 7 days that enabled complete protection of the pre-immunized mice following the CVA6 virus challenge. There were eight mutations in the genome of CVA6-A than in that of CVA6-W, possibly attributed to the virulence of CVA6 in mice. Briefly, the CVA6 infection model of the 10-day-old mice built herein, may serve as an applicable preclinical evaluation model for CVA6 antiviral drugs and vaccine study.