[Cloning and expression of 3-phosphoglycerate dehydrogenase gene and its correlative antibodies in diagnosis of autoimmune hepatitis].
OBJECTIVE
To evaluate whether the D-3-phosphoglycerate dehydrogenase (Phgdh) correlative antibodies is crucial for AIH, we cloned Phgdh cDNA and constructed plasmid, then purified and identified the immunoreactivity of the recombinant protein, and established the enzyme linked immunosorbent assay (ELISA) to detect Phgdh autoantigen correlative antibodies in diagnosis of autoimmune hepatitis.


METHODS
The constructed plasmid was transformed into E. coli. BL21(D3). This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Western blot. The ELISA with the fusion protein was established first, then, the Phgdh autoantigen correlative antibodies in serum of patients with AIH (65) and patients with PBC (122) as well as chronic hepatitis B (CHB) (56), chronic hepatitis C (CHC) (117), and normal controls (60) were detected.


RESULTS
The sequence of Phgdh autoantigen gene was the same as the sequence reported on the genebank. The fusion protein was found about 60kD strip on SDS-PAGE. Western blot analysis showed that the fusion protein had immunoreactivity. When analyzing the serum by ELISA, the immune reactivity to Phgdh was detected in 66.15% of patients with AIH, 21.42% of patients with PBC, 12.50% of patients with CHB, 6.83% of patients with CHC, and 3.30% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P less than 0.01).


CONCLUSIONS
The Phgdh cDNA is successfully cloned into E. coli BL21 (D3). The frequency of antibodies to Phgdh is much higher in patients with AIH than in patients with PBC, CHB, CHC and normal control. The antibodies to Phgdh may have utility in improved diagnosis of AIH.
XIANG
Dai-jun
DJ
Research Center for Infection and Immunity of Beijing You'an Hospital, Capital Medical University, Beijing 100069, China.
YAN
Hui-ping
HP
XIA
Qing
Q
LU
Feng
F
FENG
Xia
X
ZHAO
Yan
Y
LIU
Yan
Y
YANG
Jian-xuan
JX
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000032:analysis* / Q000235:genetics; D001324:Autoantigens / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D019694:Hepatitis B, Chronic / Q000097:blood / Q000175:diagnosis; D019693:Hepatitis, Autoimmune / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008105:Liver Cirrhosis, Biliary / Q000097:blood / Q000175:diagnosis; D008297:Male; D008875:Middle Aged; D050543:Phosphoglycerate Dehydrogenase / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055815:Young Adult
D001323:Autoantibodies; D001324:Autoantigens; D011994:Recombinant Proteins; D050543:Phosphoglycerate Dehydrogenase