ABSTRACT
On the verge of Covid-19 pandemic accompanied by the vaccination research, proposed research focuses on continuous cell lines which are actually extensively applied as essential as well as economical methods for fundamental scientific analysis, chemical rate of metabolism, analysis about degree of toxicity and formation of biological formula just like vaccines. Mouse spleen cells used are well known to survive only as primary cultures. The availability of non-cancerous long-term cell cultures is scarcely available for in vitro research studies. In order to overcome this limitation, the proposed research attempted to establish a naive, long term-continuous cell culture from Balb/c mouse. This continuous cell culture derived and established from a primary mouse spleen has the potential as a model cell culture for various applications of in vitro testing. This research can be useful for further research in the specified domain.Copyright © 2022 Wolters Kluwer Medknow Publications. All rights reserved.
ABSTRACT
The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.
ABSTRACT
Proteasome inhibitors are among the most potent classes of drugs in multiple myeloma treatment. One of the main challenges in myeloma therapy is acquired resistance to drugs. Several theories have been proposed to describe the mechanisms responsible for resistance to the most commonly used proteasome inhibitors bortezomib and carfilzomib. This study aimed to describe functional differences between sensitive myeloma cells (MM1S WT) and their daughter cell lines resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines. Bortezomib- and carfilzomib-resistant cell lines were successfully generated by continuous exposure to the drugs. When exposed to different drugs than during the resistance generation period, MM1S/R BTZ cells showed cross-resistance to carfilzomib, whereas MM1S/R CFZ cells were similarly sensitive to bortezomib as MM1S WT cells. Following proteomic profiling, unsupervised principal component analysis revealed that the MM1S/R BTZ and MM1S/R CFZ cell lines differed significantly from the MM1S WT cell line and from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons - MM1S WT vs. MM1S/R CFZ and MM1S WT vs. MM1S/R BTZ. However, important differences were present in the statistical significance of particular pathways. Key alterations included the ubiquitin-proteasome system, metabolic pathways responsible for redox homeostasis and the unfolded protein response. In functional studies, both drugs continued to reduce chymotrypsin-like proteasome activity in resistant cells. However, the baseline activity of all three catalytic domains of the proteasome was higher in the resistant cells. Differences in generation of reactive oxygen species were identified in MM1S/R BTZ (decreased) and MM1S/CFZ cells (increased) in comparison to MM1S WT cells. Both baseline and drug-induced activity of the unfolded protein response were higher in resistant cells than in MM1S WT cells and included all three arms of this pathway: IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK). In conclusion, contrary to some previous reports, resistant MM1S cells show upregulation of unfolded protein response activity, reflecting the heterogeneity of multiple myeloma and prompting further studies on the role of this pathway in resistance to proteasome inhibitors.