Anti-scirr69 Monoclonal Antibody Preparation And The Scirr69 Endoplasmic Reticulum Stress The Relationship Between A Preliminary Study

To screen out up-regulated genes after spinal cord injury (SCI) by subtractive hybridization, we had identified a novel EST (EST-69). Judged from its deduced amino acid sequences, the novel gene was predicted to code for a transcription factor of bZIP family. In the previous research, the complete cDNA sequence was obtained by 5'and 3'RACE-PCR and the novel protein molecule with 521 amino acids was designated as scirr69 (spinal cord injury repair related gene No. 69) on GenBank (accession No. AY546001). Bioinformatics analyses show that this molecule is a transmembrane bZIP transcriptional activator, which locates at rat chromosome 4q22 and exhibits extensive similarity to transcription factors belonging to the CREB/ATF family. SCIRR69 was confirmed to function as transcriptional activator in vivo and in vitro, and 60 amino acids at N-terminal accounted for the activation ability.It is prerequisite to prepare monoclonal antibodies for the further research. We predicted the antigen epitopes with potential phosphorylation sites in SCIRR69. According to the results, two peptides which were designated as S1 (pep5-22aa with phosphorylated Ser19) and S2 (pep86-101aa with phosphorylated Ser93) were synthesized commercially and linked to carrier proteins to immunize the BALB/c mice and characterize the hybridoma. In this study we established 18 hybridoma cell strains which can stably secrete mAbs against S1 or S2. The sole of the hybridoma secretes mAb against the phosphorylated S1, which was designated 1H7G5. With this mAb, full-length but not cleaved SCIRR69 could be detected, which indicated the phosphorylation status may be different during the activation process. For all, crosstalk between Nucleolin and the phosphorylated peptide S1 takes place. Among the other 17 mAbs, we choose 4B4D3D6 to identify the target molecule with WB, IFC, IP and mass spectrum for its high sensitivity. The results indicated this mAb recognized the native and denatured, full-length and cleaved SCIRR69.Due to the similarity of SCIRR69 with other ER stress transducer such as ATF6 and oasis, we investigated the expression and location mode of SCIRR69 in the presence of ER stress inducer, thapsigargin or tunicamycin, with mAb 4B4D3D6. The WB results indicated that Tg is the optimum stimulus to augment the expression of full-length and cleaved SCIRR69 in the extract of PC12 cells. IFC results showed us the location of SCIRR69 was mainly in cytoplasm of PC12 cells. When the transfected COS-7 cells were treated with Tg, part of SCIRR69 molecules transloacted into nucleus. While DTT was added into the medium, SCIRR69 mainly shifted to the polar of the cell. This staining pattern was consistent with the expression pattern of green fluorescence protein (GFP) fused SCIRR69.GRP78/BiP is an important molecule chaperone in endoplastic reticulum and activated by ER stress at the transcriptional level. The rat Grp78 promoter is a well characterized promoter system where the localization of various cis-acting regulatory elements and trans-acting factors, such as ATF6 and OASIS, contributing to both the basal and ER stress-inducible activities of the promoter has been identified. The rat Grp78 promoter contains a cAMP responsive element (CRE) and three ER stress response elements (ERSEs) conserved among Grp promoters. The LUC reporter genes were then constructed by subcloning the rat Grp78 promoter subfragment spanning -169 to -29, -300 to -29 and -457 to-29 with CRE and/or ERSEs generated by PCR into the Kpnâ… and Hindâ…¢sites of the basic luciferase vector PGL3 (Promega, Madison, WI). Utilizing the dual luciferase reporter system, no obviously increased transcription activity with SCIRR69 was detected in any promoter sequence. This result indicated the target gene of SCIRR69 is different with other transcription factors of ATF/CREB family.