| Objective:(1)The FH gene fragment was amplified by polymerase chain reactions(PCR) and inserted into prokaryotic expression vector PGEX-4T-2.The recombinant plasmid PGEX-4T-2/FH(400-510) was transformed into E.oliBL21(DE3) to be expressed. The FH fusion protein was purified with GST-glutathione affinity column and acted as an antigen to immunize the BALB/c mouse to obtain the antibody. The antibody was characterized by western blot and analysis the levels of FH in various organization. In the rat liver, FH protein complex was investigated with co-immunoprecipitation.(2) Three recombinant plasmid PGEX-4T-2/MBD1(383-455), PMAL-C5X/MBD1(227-300), PMAL-C5X/MBD1(1-70) was constructed, which was therefore transduced into E.coli BL21cells) to be expression. The fusion protein was purified to be used as an antigen to immunize the BALB/c mouse to obtain the antibody. The antibody was characterized by western blot and immunoprecipitation.Methods:(1) Expression purification of human FH recombinant protein and preparation of the antibody:The segment (1200-1530bp) from human FH gene was chose to be target gene by software analysis,which encodes110amino acid with high homogeneity between mouse and human and good antigenicity. The target segment was amplified from recombinant plasmid PCMV6-AC/FH by polymerase chain reaction (PCR) and cloned into the vector PGEX-4T-2for the construction of a recombinant plasmid PGEX-4T-2/FH(400-510), which was identified by PCR and sequenced. The recombinant plasmid transduced into E.coli BL21cells to be expressed. The DE3after IPTG induction was dissolved and centrifuged. Analyzing the exist position by SDS-PAGE. The supernatant was purified with GST-glutathione affinity column. The precipitation solubilized in the presence of8mol/L urea was dialyzed with renaturation solutions so that folding process can be initiated. Purified protein was identified by SDS-PAGE and western blot. The protein concentration was confirmed by Bradford protein quantitative determination kit protein concentration. The FH fusion protein acted as an antigen to immunize the BALB/c mouse to prepare antiserum.(2) Testing the function of antibody and looking for interacting proteins of FH.The newly developed polyclonal antibody was characterized by western blot. Analyzing the distribution of FH in rat brain, heart and the liver. In the rat liver.The interactional proteins of FH was investigated by co-immunoprecipitation. Protein mass spectrometry analyzed the interactional proteins of FH.(3) Expression,purification of mouse MBD1recombinant protein and preparation of the antibody:Choose the1-70aa,227-300aa and383-455aa peptides from MBD1as target peptides by structural analysis of MBD1. The target segment was amplified from recombinant plasmid PCMV6-AC/MBD1by polymerase chain reactions(PCR) and cloned, inserted into vectors----PGEX-4T-2and PMAL-C5X to construct recombinant plasmids: PGEX-4T-2/MBD1(383-455), PMAL-C5X/MBD1(227-300), PMAL-C5X/MBD1(1-70), which were identified by PCR and sequenced. The recombinant plasmid was transduced into E.coli BL21cells to be expressed. The fusion proteins was purified with affinity column and identified by SDS-PAGE and western blot. The fusion proteins was used as an antigen to immunize the BALB/c mouse for preparing antiserum.The anti-MBD1antibody was used to identify the MBD1from human and mouse by western blot. The function of anti-MBD1antibody was investigated by co-immunoprecipitation with mouse brain.Results:(1) The PCR amplification product is about330bp and the recombinant plasmidPGEX-4T-2/FH(400-510) was confirmed that the target gene was inserted by PCR and sequenced. Fusion protein GST-FH (400-510) was expressed by IPTG induction the DE3containing PGEX-4t-2/FH (400-510) and purified with GST-glutathione affinity column. The fusion protein is analyzed by SDS-PAGE and western blot. The results showed that fusion protein is about36kD similar to the deduced value.The western blot show that the FH endogenous proteins in rats can be recognized by FH antibody, the specific bands is around55KD.The level of FH in rat liver and heart is far higher than the brain. Protein mass spectrometry result showthat the interacting proteins of FH includes the enzymes from Krebs cycle, urea cycle and fibronectin.(2) The PCR amplification products are about210bp. We confirmed the recombinant plasmids PGEX-4T-2/MBD1(383-455), PMAL-C5X/MBD1(227-300), PMAL-C5X/MBD1(1-70) contains the purpose gene by PCR and sequenced. Fusion protein was expressed by IPTG induction the DE3Containing recombinant plasmids and purified with GST-glutathione affinity column or Amy lose affinity column. The fusion protein GST-MBD1(383-455) was analyzed by SDS-PAGE and western blot. The results showed that fusion protein is about36kD similar to the deduced value.There is an other band which is around26KD similar to the size of GST. The fusion protein MBP-MBD1(227-300), MBP-MBD1(1-70) were analyzed by SDS-PAGE and western blot. The results showed that fusion protein is about50kD similar to the deduced value.The Western blot show that the MBD1endogenous proteins in mouse and human can be recognized by MBD1antibody, the specific bands around70KD.The MBD1antibody can be used for co-immunoprecipitation.Conclusions:(1) The recombinant plasmids are constructed successfully and the target protein of FH and MBD1are expressed in E.coli BL21(DE3) and purified successfully;(2)The purified fusion protein could induce the BALB/c mouse to produce high titer specific polyclonal antibodies;(3) MBD1endogenous proteins of mouse and human can be recognized by MBD1antibody and would be used for co-immunoprecipitation.(4) FH has higher level of expression in liver and heart than brain;(5)Some enzymes from The Krebs cycle and the urea cycle and fibronectin interact witl FH. |
