| Objective: In this study,3 months,12 months and 20 months old SPF BALB/c mice were used as the research object.The characteristics of BCR H-CDR3 repertoire in mice bone marrow and spleen B cells and spleen memory B cells were analyzed by high-throughput sequencing technology.The characteristics of the repertoire were used to explore the dynamic changes of BCR H-CDR3 repertoire in B cells and memory B cells of mice at different months,and provide basic data and new research ideas for immune system aging research.Methods:1.Take B cells from bone marrow and spleen and memory B cells of spleen tissue of BALB/c mice at 3,12,and 20 months old respectively.The number of samples was:mice bone marrow tissue B cells 5 samples per month,mice spleen tissue B cells 5samples per month,spleen memory B cells 3 months per month.2.Tissue sections were made to observe the morphological differences of spleen tissue structure in 3,12,and 20 months old mice(3 per month).3.The spleen memory B cells of 3,12 and 20 months old mice were sorted by the German Milteny biotec magnetic beads sorting kit,and the purity of the sorting was identified by flow cytometry.4.The DNA in each sample was separately extracted using a QIAGEN gene extraction kit,and the purity was identified by a spectrophotometer.Each sample was constructed by multiplex PCR to construct a mice BCR H-CDR3 repertoire,and the desired target band(100-200bp)was separated by agarose gel electrophoresis and tapped and recovered.The BCR H-CDR3 repertoire was sequenced using high-throughput sequencing technology.5.The data obtained after sequencing each sample was uploaded to the IMGT(http://www,imgt.org)database to analyze the composition and characteristics of the BCR H-CDR3 repertoire sequence.(1)Analyze the diversity of CDR3 using The inverse Simpson’s diversity index(1/DS);(2)Analysis of the access to the V,D,and J gene families of the BCR H-CDR3 repertoire;(3)Analysis of the access and length distribution of AA;(4)Analysis of unique AA overlapping sequences in the CDR3 repertoire.Results:1.The white pulp of the spleen tissue of the 3 month old mice was dense.With the increase of the month of the spleen,the structure of the white pulp became irregular.2.BCR H-CDR3 repertoire feature analysis:2.1.Sequencing analysis results of B cells in bone marrow of mice at different ages:(1)BCR H-CDR3 repertoire functional unique sequence/total sequence number of bone marrow B cells in different months old mice M3BM1:470417/3628039;M12BM1:196523/1421157;M20BM1:242712/1487032M3BM2:518486/4184188;M12BM2:156116/1464383;M20BM2:200002/1310079M3BM3:381821/764189;M12BM3:64755/500687;M20BM3:120696/557000M3BM4:177963/1348026;M12BM4:183714/1101831;M20BM4:76015/426278M3BM5:178321/1293633;M12BM5:169852/1654244;M20BM5:67526/310777(2)The BCR H-CDR3 repertoire of bone marrow B cells in different months of mice was collected with high frequency IGHV1-14,IGHV1-4,IGHV14-1,IGHV1-50,IGHV1-56,IGHV1-64,Statistical analysis showed that the IGHV14-1 gene family has statistical differences;In the IGHV-IGHJ gene dominant pairing,there were statistical differences in the pairing of IGHV14-1-IGHJ3 and IGHV1-50-IGHJ4genes;The results of cluster analysis showed that the pairing of IGHV-IGHJ genes at12 months and 20 months was the closest,and the difference in 3 months was far.(3)The length of the amino acid sequence of the BCR H-CDR3 repertion in the bone marrow B cells of different months old mice is a Gaussian distribution with 11-12 amino acids,and the high frequency uses the hydrophobic amino acid represented by tyrosine(Y).There was no statistical difference in amino acid access.(4)The BCR H-CDR3 repertoire diversity(1/DS)of bone marrow B cells in different months of mice was not statistical different;The number of unique amino acids shared by mice of different ages is 29,742;The top 10 high-frequency clonal amplified amino acid sequences almost all have highly conserved amino acid motifs "WYFDV" and "YAMDY".2.2.Sequencing analysis of B cells in spleen of mice at different ages:(1)BCR H-CDR3 repertoire functional unique sequence/ total sequence number of spleen B cells in different months old mice M3S1:778821/4490891;M12S1:101368/1038593;M20S1:134395/1290012M3S2:676115/3986587;M12S2:280350/1352262;M20S2:206742/ 1651382M3S3:631673/4388235;M12S3:225851/1373556;M20S3:125701/532054M3S4:601045/3909490;M12S4:265041/1536963;M20S4:172405/995363M3S5:565442/2667877;M12S5:207727/1489695;M20S5:156198/575928(2)The BCR H-CDR3 repertoire of spleen B cells in different months of mice was collected with high frequency IGHV1-14,IGHV14-1,IGHV1-50,IGHV1-4,IGHV1-64,and only IGHV1-4 gene access was statistically different;In the accession of IGHD gene,IGHD3-3 and IGHD6-3 were statistically different with age changes;There was a statistical difference in the change of IGHJ2 gene with age.The results of cluster analysis showed that the distribution of IGHV-IGHJ gene pairing at 12 months and 20 months was the closest,and the difference in 3 months was far.(3)The length of the amino acid sequence of the BCR H-CDR3 repertion in the spleen B cells of different months old mice is a Gaussian distribution with 11-12 amino acids,and the high frequency uses the hydrophobic amino acid represented by tyrosine(Y).Among them,asparagine(N)and isoleucine(I)showed statistical differences with age changes.(5)The BCR H-CDR3 repertoire diversity(1/DS)analysis of spleen B cells in different age groups showed statistically significant differences in repertoire diversity as mice age changed;The number of unique amino acids common to mice is 46,052;The top 10high-frequency clonal amplified amino acid sequences almost all have highly conserved amino acid motifs "WYFDV" and "YAMDY".2.3.Spleen memory B cell sequencing and analysis results of mice at different ages :(1)BCR H-CDR3 repertoire functional unique sequence/total sequence number of spleen memory B cells in different months old mice M3S7:91031/1083120;M12S7:135623/725178;M20S7:22758/197852M3S8:12387/46554;M12S8:167897/869812;M20S8:68944/716833M3S9:27825/182002;M12S9:11819/76714;M20S9:6261/56436(4)The BCR H-CDR3 repertoire of spleen memory B cells in different months of mice was collected with high frequency IGHV14-1,IGHV1-50,IGHV1-14,IGHV1-64,IGHV1-4.Among them,and only IGHV1-4 gene access was statistically different;In IGHD gene access,IGHD2-3,IGHD3-1,IGHD3-3,and IGHD3-2 genes were statistically different as the age of mice changed.The analysis of IGHV-IGHJ gene pairing in different age mice showed that the same high frequency advantage paired with IGHV1-50-IGHJ1 gene.The results of cluster analysis showed that the distribution of IGHV-IGHJ gene pairing at 3 months and 12 months was the closest,and the difference in 3 months was far.(5)The amino acid sequence of the BCR H-CDR3 repertion in the spleen memory B cells of different months old mice is a Gaussian distribution with 11-12 amino acids,and the high frequency uses the hydrophobic amino acid represented by tyrosine(Y).Among them,asparagine(N)showed statistical differences with age changes.(6)The BCR H-CDR3 repertoire diversity(1/DS)of spleen memory B cells in different months of mice was not statistical different.The number of unique amino acids common to mice is 2474.The top 10 high-frequency clonal amplified amino acid sequences almost all have highly conserved amino acid motifs "WSFDV" and "WYFDV".Conclusion:1.Irregularity of white medullary structure of spleen tissue with increasing age of mice.2.Age-related decline in the diversity of BCR H-CDR3 repertoire in mice spleen B cells;Different ages of mice have different access to the IGHV gene family in the BCR H-CDR3 repertoire of bone marrow and spleen B cells and spleen memory B cells,and the IGHJ gene was similarly used.3.As the age of mice increases,the length of amino acids increases gradually,but the amino acid usage changes little and there are highly conserved amino acid sequences. |
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