| T cell receptors play an important role in immune response and immune regulation. Since 1980s, genes of TCR have been cloned gradually, the relationship between TCR and acquired immunity, autoimmune disease, lymphocytic malignancy, virus have been extensively researched,bringing a glimmer of hope for the disease control and prevention. At present, rapid progress was made in researching TCR of human and mouse, but the structure and function of porcine TCR can't find more literatures, which delay the development of veterinary immunology and lead to animal disease control and prevention without well guiding of modern immunology theory. Based on the sequence TCRαchain,βchain deposited in GenBank, porcine TCRαchain,βchain were cloned on the domestic for the first time, the structure and function were deeply analyzed by relative bioinformatics software, then analyzed and classified the allele polymorphism among sequence deposited in GenBank and sequence cloned by our lab in order to study their immune mechanism. Meanwhile, one of representative TCRαchain were cloned into prokaryotic expression vector, then induced expression in recombinant Escherichia.coli BL21, high purity of recombinant protein was obtained. Result as follows:1. Depended on phylogenetic analysis and gene rearrangement to design primmer, and RT-PCR technology were used, porcine TCRαchain,βchain were cloned from porcine mesentery lymphocyte on the domestic animals for the first time. After sequence alignment among TCRαchain,βchain sequence deposited on GenBank, 12 TCRαchain sequence (pTRAV4-15)were cloned with the primers designed on the open reading frame based on the sequence of AB087988, and shared 91.5%～98.5% homology. As well, 3 TCRαchain sequence (pTRAV1～3)were cloned with the primers designed on the open reading frame based on the sequence of AB087990, and shared 98.5%～99.6% homology. 16 TCRβchain sequence were cloned with the primers designed on the open reading frame based on the sequence of AB079529, and shared 98.5%～99.6% homology. Cloned sequence were confirmed as objective sequence by alignment with model chain and BLAST on NCBI web, and deposited on Genbank, 15 porcine TCRαcDNA sequence pTRA1～15 assigned accession number as FJ944028～FJ944042.2. Based on bioinformatics software, the structure, homology and polymorphism of TCRα,βchain and deducted coding protein were analyzed. With some bioinformatics software such as ClustalW, DNAStar, MEGA, DNAMAN and website such as NCBI (http://www.ncbi.nlm.nih.gov/),EXPASY (http://www.expasy.ch/) and IMGT/V-QUEST (http://imgt.cines.fr/IMGT_vquest/vquest), The genes of porcine TCRα,βchain were analyzed on bioinformatics and polymorphism, V domain and J domain are the main polymorphism domain, among which CDR3 has highly polymorphism. Alignment with sequences deposited on GenBank, there is some regularity of polymorphism in V domain. Alignment among TCRαchain sequence cloned by our lab, pTRAV 1 with 2 and 3 share 98.5% and 99.6% homology, so are bracketed together;pTRAV5 with pTRA7, pTRA9 and pTRA13 share 97.7%, 99.6% and 98.5% homology, so are bracketed together; compared with pTRAV4 and 12, share 98.9% homology, so are bracketed together; with pTRAV6 and 10, share 98.9% homology, so are bracketed together; with pTRAV11 and 14, share 99.3% homology, so are bracketed together; pTRAV8 are bracketed alone; Alignment among sequence deposited on NCBI, V domain share lower homology, the gene of TCRαchain should be firstly cloned by our lab. Homology alignment analysis among 15 TCRβchain amino acid sequence show that CDR3 domain is obviously polymorphic due to recognizing polymorphic antigen. Alignment between pTRBV16 and pTRBV1～15 on nucleotide level, pTRBV16 maybe a pseudogene due to lack of 50～302 loci which is variable region.3. According to categorizing method of Yamamoto, alignment between the V region of porcine TCRαchain and the V region of human TCRαchain, the homology between pTRAV1～3 and human TRAV16*01 above 80%, grouped to one group; but the homology of pTRAV4～14 were below 75%. pTRAV4, 5, 6, 8～14 and human TRAV18*01 share 71.91%～73.41% homology; pTRAV1～3 and human TRAV16*01 share 81.99%～83.91% homology; pTRAV7 and human TRAV8-4*07, TRAV18*01 share 62.37%, 54.92% homology respectively. Sequence homology analysis among V domain of porcine TCRαchain deposited on NCBI, V domain of porcine TCRαchain cloned by our lab and V domain of human TCRαchain, using MEGA to construct Neighbour-joining tree (the bootstrap significance values 10,000 replicates), found that pTRAV 4～14 and TRAV18*01 were classified to one group, among which pTRAV 14, Va15 and pTRAV11 were classified to one subgroup, but pTRAV 4～14 and Va14 were classified one subgroup. pTRAV1～3, Va27 and human TRAV16*01 were classified to one group.TCRβchain and the V region of human TCRβchain on web IMGT/V-QUEST, the homology between pTRBV1～15 and human TRBV15*01 or TRBV15*02 above 75%, classified to one group. Sequence homology analysis between V domain of porcine TCRβchain cloned by our lab and V domain of human TCRβchain on web IMGT/V-QUEST, using MEGA to construct Neighbour-joining tree, found that pTRBV1～15 and TRBV15*01 were classified to one group. Among which TRBV4 were classified to one group alone, pTRBV 1～3, 5～15 classified to one group.4. The representative porcine TCRαchain, pTRAV1 and pTRAV8 were modified, with 687bp and 675bp respectively, and cloned into prokaryotic expression vector: pET30a and pET28a, then the product induced expression in BL21 by IPTG confirmed by SDS-PAGE, Western-blot. Recombination protein with highly purity and rightly architecture, 27ku and 30ku respectively, were obtained as inclusion bodies.The document on porcine TCR studies is seldom, the sequence information of TCRαchain,βchain is beneficial to the study on molecular structure, genealogical classification, and biological function of porcine TCR.
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