Auto-Antigens Targeted By Auto-Antibodies IgG on the Bone Marrow Hematopoietic Cells of the Patients with Immuno-Related Pancytopenia

Objective: To screen and identify the auto-antigens targeted by auto-antibodies IgG on the bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP).

Background: Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression.. Our previous studies have demonstrated that DC2 increased in the bone marrow of IRP patients, which promote Th0 cells to polarize to Th2 cells and cause the over-function of B lymphocytes.These B cells produce auto-antibodies that mediated BM destruction.. But the antigens that induced DC2 elevated are not clear at present, our group found several target antigens by western blot method before, including G protein coupled receptor (GPCR) 156 variants, P chain of red blood cells with band three proteins, lactoferrin and WD repeat sequences of proteins.Autoimmune diseases often have several or even dozens of target antigens, so this experiment adopts the method of cDNA expression library of serological analysis (SEREX) to sceen antigens targeted by IRP autoantibodies and further verify their antigenicity.

Materials and Methods: We established the recombinant cDNA expression libraries derived from K562 cells. We performed immunoblotting analysis of this library with sera from patients and normal healthy controls. The presumptive phage expressed autoantigen proteins were initially proposed based on the differential affinity to the sera antibodies of patients versus normal healthy controls,the plagues which were positive with IRP serum but negative with normal control serum were positive screened plagues. The insert fragment of each positive plague was sequenced. After the gene sequence confirmation, the sequence of FTL gene, TRMT112 gene and TRAPPC gene were amplified by polymerase chain reaction (PCR) then inserted into Peasy-E1 vector separately. Each recombinant plasmid was transformed into E.coli BL-21 and was identified by PCR and sequencing. After gene identification, the right recombinant plasmid was transformed into E.coli BL21 and the bacteria was induced by isopropyl-β-d-thiogalactoside (IPTG) to express. The expected protein was identified by Western blot and purified by ProteinIso Ni-NTA Resin. Then we coated ELISA plate with purified FTL protein and EPOR protein respectively, and detected the titer of the serum antibody of IRP patients and normal controls by indirect ELISA. Finally, the clinical index of 121 IRP patients were analysed.

Results: 1.We established the recombinant cDNA expression libraries derived from K562 cells successfully. The library has good capacity, ligation efficiency and recombination efficiency. 2. We screend 11 positive plagues by immunoblotting, the seven of them were sequenced and blasted successfully. 3.3 fusion protein was expressed and confirmed by Western blot. The purified protein was obtained by ProteinIso Ni-NTA Resin. 4. The titer of antibody against the FTL and EPOR proteins was higher in untreated IRP than in recovered IRP patients and in normal control.

Conclusion: We screened 7 proteins that maybe autoantigens of IRP by SEREX. Furthermore, we expressed and purified 3 fusion proteins. Finally we identified the antigenicity of the FTL and epor proteins.