The Regulatory Effects of Purα on PARP1 Gene Expression and its functions on DNA repair

The luciferase reporter construct, which contains a poly ADP-ribose polymerase 1 gene promoter, was transfected into U87MG cells with the Purα eukaryotic expression vector, and the activities of PARP1 promoter were assessed by luciferase assay to evaluate the regulatory effects of Purα on PARP1 gene expression. The Purα eukaryotic expression vector was transfected into U87MG cells, and the cell total RNA and protein were extracted to determine the effects of Purα on PARP1 gene expression at transcriptional and translational levels by real-time PCR and Western blotting assay. The results demonstrated that Purα can positively regulate PARP1 promoter activity and promote PARP1 gene expression both at transcriptional and translational levels. The further study illustrated that Purα can collaborate with PARP1 in the repair of damaged DNA; the results of a pull-down assay suggested that there is a physical interaction between Purα and PARP1. The overexpression of Purα can increase endogenous PARP1 expression and alleviate the expression of the DNA damage signal protein γH2AX. Above all, we believe that Purα possesses a positive regulatory effect on PARP1 gene expression and collaborates with PARP1 to repair damaged DNA. Keywords: Purα; poly ADP-ribose polymerase 1; regulation of gene expression, DNA damage, and repair.

Cloning expression and immunogenicity analysis of inhibin gene in Ye Mule Aries sheep

Background Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INHα. Following the validation of successful cloning, the pEGFP-INHα plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results The analysis of the INHα gene sequence revealed that INHα is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INHα gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INHα, which expresses the INHα gene elicited immune response and generatigeneration on of anti-INHα antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INHα and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INHα was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INHα on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.

Structural predication and antigenic analysis of Toxoplasma gondii ROP20

Toxoplasma gondii infects almost all the warm-blooded animals. ROP20 protein is expressed in the rhoptry of Toxoplasma gondii. In this study, the secondary structure of ROP20 was analyzed using SMART software. We constructed and analyzed the 3D model of ROP20 protein using SWISS-MODEL online procedure and Visual Molecular Dynamics (VMD) software. The structure analysis fully indicated that ROP20 protein is an important member of the ROP family. Furthermore, We used DNASTAR software and Epitope Database online service to analyze liner-B cell epitopes and T-cell epitopes of ROP20 protein. All the analysis results of ROP20 protein can provide positive information on treatment and vaccine for toxoplasmosis. Moreover, ROP20 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-C1-ROP20) was constructed in the following study. After restriction enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result indicated that the recombinant plasmid could transcribe successfully in HEK 293-T cell. The results of western blotting indicated the expressed proteins can be recognized by anti-STAg mouse sera.