Purification And Identification Of Recombinant PDC-E2 And Preliminary Study Of Relationship Between HLA Genotypes And Susceptibility To PBC In North Chinese

Primary biliary cirrhosis (PBC) is characterized by an immune mediated, irreversible destruction of the small intrahepatic bile ducts leading to progressive liver cirrhosis and frequently to liver failure. In recent years, more attention has been paid for PBC in our country, and the incidence of detected cases of PBC has been increasing. Because of lacking effective therapeutic measures, so it is necessary to reinforce the study of pathogenesis of PBC.The course of PBC is variable and an early diagnosis is desirable to identify individuals with rapidly progressing disease, to initiate adequate therapeutic measures and to predict the timing of liver transplantation. AMA (anti-M2) are an exclusive serologic hallmark of PBC. AMA in PBC are directed to the three ketoacid dehydrogenase complexes, and predominantly to E2 subunit of pyruvate dehydrogenase complex (PDC-E2). PDC-E2 is considered to be the most possible target antigen that mediated the damage of the intrahepatic bile ducts. Autoantibodies to PDC-E2 can be detected in sera of 95% of PBC patients. So purified PDC-E2 will be helpful for further study of pathogenesis of PBC and diagnosis of PBC. In this study, we purified the recombinant PDC-E2 expressed in E.coli. by using immune affinity chromatography method and identified its immune reactivity with sera of PBC patients by using Western blot analyses and ELISA. The results showed that the positive rate of autoantibodies against purified PDC-E2 in sera of PBC patients is 100%, while that in sera of patients with other liver diseases and healthy people is zero, which suggest that the purified PDC-E2 could highly specifically bind the autoantibodies in the sera of PBC patients. After using purified PDC-E2, the test procedure of our first generation of ELISA kit was simplified and false positive result was avoided.As that of many other autoimmune diseases, genetic factors were considered to play an important role in pathogenesis of PBC. Some of MHC class II molecules, especially HLA-DR8, were reported to be associated with susceptibility to PBC, but results from different population are not unanimous and there has been no data about north Chinese people. The HLA-DR and DQ gene of 67 north Chinese patients with PBC and controlled 231 health people, was genotyped by using forward DNA hybridization Microarray technique and SSP-PCR method in this study. The frequency of DRB1 * 08 was 23.9% in PBC patient group, much higher than that (6.93%) in health group (RR=4.22, P=0.000) and that (8.7%) in disease control group (P=0.038);HLA-DRB1 * (^ DRB3 (DR52) > DQB1 * 03 and DQB1 * 06 has potential role in susceptibility to PBC. The results suggested that DR8 were associated with PBC as a risk factor in north Chinese people. Further study in this respect will be helpful for understanding of etiology and the prediction and diagnosis of this disease.