Prokaryotic Expression And Purification Of IdpA And Preparation Of Its Monoclonal Antibody

The phytoplasma has no cell wall, which is peridiumed by three-layer membrane, showing the polymorphism. The phytoplasma is still not in vitro cultured. The cell membranes of phytoplasma are directly contacted with the host plant and body cells of insect.The phytoplasma membrane proteins may be involved in the interaction with the host-pathogen, therefore it is great significance to study membrane proteins of phytoplasma, which may explain the interpretation of phytoplasma evolution, pathogenicity and host interactions. Immunodominant membrane protein A (IdpA) is one of the most important member of the phytoplasma membrane proteins. There are more than ten genes encoding immune membrane proteins. Researchs on phytoplasma gene have laid a basis to carry out relevant research by using genetic engineering means for us. There are monoclonal antibodies of immunodominant membrance protein and the gene chip by using molecular biology and immunology, and the establishment of new detection methods provides a better way to the detection of phycoplasma.In this study, we constructed prokaryotic expression vector by using molecular biology tools, the phytoplasma immunodominant membrance protein A (IdpA) was induced to expressed by the induecr IPTG, and the6X His tag was introduced in the fusion protein. The IdpA protein was purified by Ni-NTA His-Bind affinity chromotraphy. We immuned BALA/C mice with the purified protein to produce the monoclonal antibody of phytoplasma immunodominant membrance protein A IdpA, which laid a foundation for produing a fast, convenient and high-throught protein chip.The IdpA gene fragment was amplified by polymerase chain reaction(PCR) from the recombinant plasmid pMD18-T-IdpA. The PCR has same fragment size with the target gene IdpA(864bp) in750-1000bp. The IdpA gene was cloned into prokaryotic expression vector pET-28a(+) by enzyme digestion and connection, and the recombinant plasmid was transformed into E.coli BL21(DE3). The assay of PCR and double enzyme digestion confirmed that the recombinant plasmid pET-28a(+)-IdpA was transformed into prokaryotic expression vector successfully. Plate culture of recombinant strains was used to screen positive clones, and the growth curve of the positive recombinant strain was determined. IPTG was used in the logarithmic growth phase to induce the expression of the target protein IdpA when the value of ODgoo was at1.0. The expression of IdpA was primarily identified by SDS-PAGE. As fusion protein was matched with6X His-Tag, so the antibody with HRP-labeled anti-His was used to indicate that the fusion protein IdpA can be expressed with6X His-Tag by Western blotting. At last, the stable recombinant strain was screened successfully. IdpA protein was expressed in the form of inclusion body by soluble analysis, we obtained the target protein by using modified purification with Ni-NTA His-Bind affinity chromatography and dialysis renaturation method. The purity of IdpA protein was more than90%, the concentration of target protein was approximately0.8mg/ml after the dialysis and ultrafiltration tube. BALB/c mice was Immuned with the purified protein, and detected the tail blood by ELISA after three times immunization. The anti-serum titer was higher than1:320000. Western blotting showed that the antiserum was specific. Three days before the cell fusion, we did booster immunization through intravenous, then fused the mice spleen cells and SP2/0cells of logarithmic growth phase with the PEG4000with the concentration of50%. We detected titer of the cell supernatant by ELISA and screened the positive clones. After two rounds screening, three monoclonal cell lines which secreted stablely IdpA protein antibody were obtained.