Primary Analysis Of CDR3 Spectratyping And Molecular Features Of TCR Alfa And Beta Chains In CD8~+T Cells Of Swine Immunized By CSFV-C Strain Vaccine

Antigen-specific T cell receptor(TCR)cloning studies have been widely used in the diagnosis and treatment of human diseases,but less in animal diseases,especially swine fever.In this study,the pigs were stimulated using the classical swine fever virus-C strain(CSFV-C strain)vaccine to produce the immune response against the swine fever virus,and the TCR gene family,which is specific for the swine fever antigen,were screened by analyzing the peripheral blood CD8~+T cells TCR complementarity determining region 3(CDR3)spectrum then further screening was performed by secondary stimulation.The CDR3 of the TCR families,which showed explicit monoclonal or oligoclonal amplification,were cloned into the T vector by PCR.Several clones were sequenced and the same gene sequences and conserved motifs were found in these CDR3 clones.The results showed that there were monoclonal and oligoclonal proliferation of TCR families in peripheral blood CD8~+T cells after immunization with swine fever,and showed regular changes and conservative amino acid sequences.This study showed that monoclonal and oligoclonal expansion of the TCR families was associated with the identification of classical swine fever virus antigen peptides,and could provide theoretical support for the development of classical swine fever virus T cell epitope vaccine.This study has made the following progress:1.The CDR3 spectratypes of??TCR in CD8~+T cells were analyzed after the first immunization successfully.19 TRAV and 20 TRBV gene families of CD8~+T cells were amplified by RT-PCR,and the spectral distribution and sequence characteristics of CDR3 were analyzed by gene scanning-spectratyping technique.The results showed that almost all CDR3 regions of TRAV/BV families showed typical Gaussian distribution before the first immunization.Most families contained more than 8 peaks with different fluorescence intensities,different lengths and sequences,indicating that the CD8~+T cell??TCRs were highly diverse before the first immunization.On the third day after immunization,some TCR families began to show clonal expansion and this clonal expansion was maintained for a certain period of time,and then most clonal families returned to the Gaussian distribution on the 15th day post immunization,suggesting that the clonal changes of CD8~+T cells were associated with swine fever virus infection.2.The changes of serum antibody titer after the second immunization were analyzed.Res-ults showed that the antibody of the second immunization had a higher titer and lasted for a longer ti-me than the first immunization,indicating that the body has produced an effective immune mem-ory towards the C-strain vaccine and the body's immune system produced a highly effective i-mmune response to the second immunization.3.The spectral changes of??TCR in CD8~+T cells were analyzed successfully after the second immunization.Similar to the first immunization,most of the CDR3 spectratypes of TRAV/BV families were typical Gaussian distribution before the second immunization;the difference was that the clonally expanded TCR families of CD8~+T cells appeared earlier than the first immunization,and the RI value of the clone peak of TCR family was significantly stronger than that of the first immunization.At the same time,AV8-3S and AV22 in P1 pig,AV4S in P2 pig,DV1 and BV15S in P4 pig showed clonal expansions both at the first immunization and the second immunization,demonstrating that after the second immunization,classical swine fever virus-specific memory T cells were activated and proliferated.4.The sequence characteristics of CDR3 of TRAV and TRBV families showing clonal expansions were analyzed.The CDR3 of the clonally expanded TCR gene families was amplified by PCR and the bioinformatics analysis was performed after the cloning and sequencing.Sequence analysis showed that most of the CDR3 from clonally expanded families had conserved amino acid sequences.For the clonally expanded TRAV families,the"KL"amino acid motif appears in four of the five families,and each family also has conservative amino acid motif;for the clonally expanded TRBV families,the"QY"amino acid motif appears in five of the seven families,and"GG"appears in six of the seven families.In addition,high level of expression of specific amino acid sequences in clonally expanded AV22,AV4,BV27 and BV15 gene families were observed,and those amino acid sequences were considered to be the dominant expression sequences.The four families which also showed clonal expansiosn at another time were re-sequenced,and these dominantly expressed sequences indeed showed high frequency of expansion.In contrast,before the immunization,the four families showed heterogeneity expression,and no dominant expression sequence was found.These results suggest that these dominantly expressed conserved amino acid motifs are associated with the specific identification of swine fever virus.