Irritable bowel syndrome detection of serum anti-Hu antibody and its clinical significance in patients with
|School||Beijing Union Medical College|
|Keywords||HuD protein Plasmid vector Procaryon expression system GST fused protein Paraneoplastic syndromeanti-Hu antibody anti-enteric neuronal antibody irritable bowel syndrome (IBS) Biomarker|
BackgroundHuD protein, as an important member of ELAVL family, is specificly distributed in nervous tissue. It is important for growth, development and differentiation of neurons. HuD protein functions by binding3’-UTR ARE element of mRNA in neuron, which might promote the stabilization and translocation efficiency of mRNA. It is proved that there is some certain relationship between HuD low expression and Hirschsprung disease. The polyclonal antibody, anti-Hu antibody, plays a role in pathogenesis of paraneoplastic syndrome and pseudo-intestinal obstruction. To obtain purified HuD protein with natural bio-immunol reactivity is of great value to study its biological function and understand the role of anti-Hu antibody in the pathogenesis of some diseases.ObjectiveTo build the recombinant plasmid of mouse HuD DNA vector with GST tag, and express it by procaryon expression system. Purify and identify the HuD protein.MethodsExtracting the total mRNA in the brain of mouse and RT-PCR with designed primers to obtain special DNA segment which encodes HuD protein. A recombinant plasmid of GST-HuD was expressed in procaryon expression system. GST-HuD fused protein was purified with the help of GST-tag following a cut off of GST tag. Finally the HuD protein of mouse was obtained.ResultsObtained purified GST-HuD fused protein and HuD of mouse, which maintained its natural bio-immunol reactivity.ConclusionsHuD protein of mouse obtained by molecular clone technique maintained its bio-immunol reactivity. It is convenience for further study on the neurobiology function of HuD and clinic significance of its polyclonal anti-Hu antibody. BackgroundAnti-Hu antibody is a kind of anti-neuron nuclear antibody. It is also called anti-neuron nuclear antibody type I. The antigen of anti-Hu antibody, HuD protein, was specially distributed in neurons. It is important for the growth, develop and differentiation of neurons. It was first reported the anti-Hu antibodies in CSF and serum of paraneoplastic syndrome (PNS) patients. And now it is widely used as a sero-biomarker for PNS, to diagnosis and prediction of prognosis. Functional bowel disorders (FBD) is diseases without any organic disease. It presents mainly sensation and movement disorders of gut. Its diagnostic criteria, Rome III, are based on symptomatology for the complication of its pathogenesis. The latest progress on FBD showed some clew that some autoantibody in the serum were related to FBD and since that, a potential new biomarker would come out. IBS is a main type of FBD, which includes IBS-D, IBS-C, IBS-M, and IBS-U. It is a common disorder with5%-20%of prevalence. The pathogenesis of IBS is not well understood. The recent research reported by Wood JD and his collogues revealed the correlation between autoanti-enteric neuronal antibodies and IBS (DDW,2010).About79%of IBS patients in their investigation are anti-enteric neuronal antibodies sero-positive, which might be of anti-Hu antibody. The role of anti-Hu antibody in IBS pathogenesis needs more study.ObjectiveTo investigate the positive ratio of anti-enteric neuronal antibodies (especially anti-Hu antibodies) in IBS group and health control group by indirect immunofluorescence, immunol blotting and ELISA technique. To explore the relationship between the antibodies (including anti-Hu antibody) and IBS (including IBS-C and IBS-D)MethodsOutpatients of IBS and health volunteer from Peking Union Medical College Hospital(PUMCH) were enrolled. And their serum samples were centrifuged and restored at-80℃. Indirect immunofluorescence was used to detect the anti-enteric neuronal antibody with the substrate of enteric submucosal plexus of guinea pig. Western blot and ELISA were used to evaluated the anti-Hu antibodies qualitatively and quantitatively.ResultsThe positive ratio of anti-enteric neuronal antibodies is51/81in the IBS group (4/6in IBS-C and47/75in IBS-D), comparing with6/86in the health control group. The positive ratio of anti-Hu antibody is7/81in the IBS group (3/6in IBS-C and4/75in IBS-D), comparing with0/86in the health control group. There are significant differences between IBS and health control groups in both anti-enteric neuronal antibody and anti-Hu antibody research.ConclusionsAnti-enteric neuronal antibodies (especially anti-Hu antibodies) might play a certain role in the pathogenesis of IBS. It should be evaluated further that anti-Hu antibody has the potential value to be a biomarker for IBS.