The Cloning And Expression Of Pig TREM2 And The Preparation Of Polyclonal Antibody For Its Extracellular Domain

Triggering Receptor Expressed on Myeloid Cells 2,a class of innate immunity receptor,plays an important role in innate host defense. to date, the study of porcine triggering receptor on myeloid cells still lie in a initiatory stage. To investigate the biological function of the trigger receptor expressed on myeloid cells 2, Using the RNA extract form Porcine alveolar macrophages as a template to amplify the full gene fragment of TREM2 verified by sequencing according to the sequence of TREM2（NM: 001256777.1） in GenBank.Then, the gene fragment of the extracellular domain of TREM2 inserted into the prokaryotic expression vector pET-28a（+）. Recombinant protein TREM2 was efficiently expressed by E.coli in the formation of insoluble inclusion bodies. Subsequently, affinity chromatography purify TREM2 protein. Using purified TREM2 as immunogen to immunize BALB / c mice, about100 ug for each one to produce antiserum, which can identify swine TREM2 molecule Therefore, in this study, an underlay was prepared to explore the function of TREM2 in pathogenic infection and illustrate role in swine respiratory disease.There are three parts in this study:1 Cloning and bioinformatics analysis Sequence of the porcine TREM2 moleculeThe effects of TREM2 molecule draws growing concern in inflammation. In particular,the inflammation caused by porcine respiratory disease is growing in our country. Its propagation fast, rapid break out, has brought great disaster to the pig industy. Therefore, the of research TREM2 molecular in porcine respiratory inflammation has great prospects.A pari of primers was designed by Primer5.0 according to reference sequence TREM2（NM₀01256777.1） in GeneBank to amplify target gene with reverse-transcripted RNA extracted from porcinealveolar macrophage. Correctness was verified by sequencing the amplified sequence. Alignmenting amplified productand encoded amino acid to reference in GenBnak. While a phylogenetic tree include pig human and mouse wasseted up toanalyze the genetic differences, hydrophilic, transmembrane domain and B cell antigen epitope prediction of TREM2 sequence in order to clarify TREM2 protein epitopes, phylogeny, development and establishment of a new polyclonal antibody detection method provides theoretical and experimental basis.2 The construction of prokaryotic expression vector, expression and purification of porcine TREM2The result of bioinformatics sequence analysis according to the cloned porcine TREM2 molecule. We selected the extracellular domain of porcine TERM2 molecules to carry on prokaryotic expression, designed primer of prokaryotic expression vector, the extracellular domain of swine TERM 2 molecule is connected to the expressed vector pET-28 a.Transformed recombinant expression vector to competent E.coli BL21（DE3）, by optimizing the conditions for inducible expression, to determine the best induced condition. SDS-PAGE and by high expression of recombinant proteins electrophoresis using the His-Trap Ni-chelating chromatography The recombinant protein was purified, the final purification of recombinant proteins by SDS-PAGE electrophoresis successfully detected.3 Preparation and Application of polyclonal antibody molecule TREM2The purified recombinant protein with Freund’s adjuvant ratio of 1:1 mixture of emulsified, A mice immunized about 100 μg, blood was collected one week after the fourth immunization, separation of serum obtained polyclonal antibody. By Western-Blot prove that porcine TREM2 prokaryotic expression of recombinant protein has good antigenicity, The results show that immunization is not only a polyclonal antibody capable of reacting with swine TREM2 molecule, pigs can also eukaryotic expression vector expressing full-length reaction TERM2（about 31 KDa）.