| Objective:With three months old GF,SPF and CL BALB/c mice as the research object,this paper analyzed the characteristics of the memory B cells BCR H-CDR3 repertoire of the mouse’s small intestine and spleen.This study preliminarily explored the relationship between intestinal microflora and BCR H-CDR3 repertoire of mouse intestinal and spleen memory B cells.This study provided reference data,new research ideas and techniques for further study on the development,differentiation,proliferation and response of B cells to intestinal microflora.Methods:1 The faeces of three months old GF,SPF and CL BALB/c mice were collected at-80 degrees.The DNA of microbial community was extracted,and of which the 16SrDNA was libraried and Illumina Solexa sequencing were carried out.EXCEL software and Draw Venn Diagram software were used to analyze the composition and characteristics of the GF,SPF and CL mice fecal microbacteria groups.(completed by Huada gene)2 The histological structure of small intestine and mesenteric lymph nodes of three months old GF,SPF and CL BALB/cmice were observed by using tissue section technique.3 The percentage of memory B cells in small intestine,spleen,bone marrow and peripheral blood of 3 months old GF,SPF and CL BALB/cmice was detected by flow cytometry.4 The memory B cells of small intestine and spleen were obtained by magnetic bead sorting technique.The purity of memory B cells was identified by flow cytometry,and the purity of B cells was more than 85%.QIAGEN kit was used to extract DNA samples of memory B cells from small intestine and spleen,and DNA samples from small intestine tissue.Firstly,the concentration and purity of each DNA sample were identified.The BCR H-CDR3 repertoire was constructed with multiple PCR in each mouse sample,and the Illumina Solexa high-throughput sequencing was carried out(completed by Adaptive Biotechnologies ImmunoSEQ,USA).5 Using Immuno SEQ software,draw Venn Diagram software and GraphPad Prism 5 software to analyze the composition of memory B cell BCR H-CDR3 repertoire in small intestine and spleen and total B cell BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL mice,including the usege of V,D,J,V-J gen pairing,the high frequency mutation of V gen,amino acid length,the usege of amino acid,sequence composition of iso-alanine,insertion and deletion of nucleotide.Thus,the diversity,cloning and specificity of CDR3 repertoire can be analyzed.Results:1 The Tag/OTU sequences number of feces sequencing in three months old GF,SPF,CL BALB/c mice.(3 mice in each group)MGF1:45554/48;MGF2:43787/56;MGF3:45086/38;MSPF1:35225/293;MSPF2:35050/288;MSPF3:35401/287;MCL1:37134/329;MCL2:36610/335;MCL3:36025/302;There was significant difference in the diversity of intestinal microflora among the three groups(P<0.05).The main expression was that CL mice had the largest number of intestinal microflora and the most abundant diversity.Among them,CL mice had more Proteus than SPF mice.But the faeces of GF mice had very little species and low abundance.2 The histological structure of intestinal and mesenteric lymph nodes of GF BALB/cmice was different from that of SPF and CL BALB/cmice.The main difference was that the small intestinal villi were diluted,the villus length was relatively long,the crypt depth was deeper,and the glandular layer and the basal layer were thinning;The primary germinal center of mesenteric lymph nodes became smaller,and there was less diffuse lymphoid tissue between lymph nodules in the superficial cortex.3 The distribution of memory B cells in bone marrow,spleen,small intestine and peripheral blood of three months old GF,SPF and CL BALB/cmice is different.(3 mice in each group)The main difference was that the proportion of memory B cells in bone marrow of GF-grade mice was significantly lower than that of SPF grade and CL group(P<0.05),while the proportion of memory B cells in spleen and peripheral blood was significantly increased(P<0.05).4 Total HTS sequence data and analysis results of BCR H-CDR3 repertoire in small intestine and spleen of three months old GF,SPF and CL BALB/cmice(27 samples).(1)Sequencing and analysis results of total B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/cmice(3 mice in each group)a.The number of unique sequences and total productive sequences of total B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/c mice(9 samples).MGF1-SI:368/456;MGF2-SI:10612/12772;MGF3-SI:636/945;MSPF1-SI:11346/14556;MSPF2-SI:5636/7639;MSPF3-SI:10614/12422;MCL1-SI:1813/2573;MCL2-SI:1826/2596;MCL3-SI:2372/3485;b.Total B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/c mice all high frequency used IGHV03-02,IGHV14-02,IGHV09-03,IGHD01-01,IGHD02-03,IGHJ04-01,and IGHJ01-01 gene.The comparison among the three groups,there was no significant difference in the gene usege of IGHD except for the gene usege of IGHV,IGHJ and V-J gene pairs.The main differences were as follows:Compared with the GF mice,the gene usege of IGHV01-09 and IGHV15-02 in SPF group and CL group were significantly reduced(P<0.05),and the gene usege of the IGHV03-06 was significantly elevated(P<0.05);Compared with GF group and SPF group,the usege of IGHV15-02 in CL group were significantly reduced(P<0.05);Compared with GF group and SPF group,the gene usege of IGHJ04-01 in CL group were significantly reduced(P<0.05).Compared with the GF grade group,the gen pairing usege of IGHV03-02-IGHJ03-01 was significantly increased in both SPF and CL groups(P<0.05).c.The amino acids usege distribution of total B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/cmice were all bell-shaped with 11-12 amino acids as the midline,and the hydrophobic amino acids represented by tyrosine were used in high frequency.The differences of BCR H-CDR3 gene pair in total B cells in small intestine among the three groups were as follows:compared with GF group and SPF group,the usege of methionine in CL group was significantly lower than that in GF group and SPF group(P<0.05).d.There was no significant difference in diversity of total B cell BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/c mice and unique amino acid sequences shared by the three groups was 37.The first 10 high frequency cloned amino acid sequences in the three groups had highly conserved amino acid motifs,namely"WYFDV",in which GF group was the most.The main differences of nucleotide insertion and deletion among the three groups were as follows:compared with the other two groups,the number of terminal deleted nucleotides in D5’ of CL group decreased significantly(P<0.05),and compared with SPF group,the total number of nucleotides inserted at the broken end of CL group increased significantly(P<0.05).The main difference of the high frequency mutation sequence of IGHV gene among the three groups was that compared with the other two groups,the mutation rate of CL group in 400-800 was significantly higher than that of the other two groups(P<0.05).(2)Sequencing and analysis results of small intestinal memory B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/cmice(3 mice in each group)a.The number of unique sequences and total productive sequences of memory B cells BCR H-CDR3 repertoire in small intestine of three months old GF,SPF and CL BALB/c mice(9 samples).MGF1-I:88/89;MGF2-I:352/371;MGF3-I:20/20;MSPF1-1:958/1004;MSPF2-1:1935/2043;MSPF3-1:1843/1949;MCL1-I:2371/2799;MCL2-I:33/35;MCL3-1:146/172;b.Memory B cells BCR H-CDR3 repertoire in small intestinal of three months old GF,SPF and CL BALB/cmice were all high frequency used IGHV14-02,IGHV03-02,IGHV09-03,IGHD01-01,IGHD02-14;IGHJ04-01-01,IGHJ03-01,IGHHJ03-01,IGHJ03-01,IGHJ03-01,IGHJ03-01,IGHJ03-01.The comparison among the three groups,there was no significant difference in the gen usege of IGHD gene except for the gen usege of IGHV,IGHJ and V-J gene pairs.The main differences were as follows:the gen usege of IGHV06-06 in both SPF and CL groups was significantly higher than that in GF group(P<0.05).Compared with GF group,the usege of IGHJ04-01 in SPF group was significantly higher(P<0.05).The gen pairing usege of IGHV03-02-IGHJ03-01 and IGHV14-02-IGHJ04-01 genes in both SPF and CL groups was significantly higher than that in GF group(P<0.05).c.The amino acids usege distribution of memory B cells BCR H-CDR3 library in small intestinal of three months old GF,SPF and CL BALB/cmice were all bell-shaped with 11-12 amino acids as the midline and the hydrophobic amino acids represented by tyrosine were used at high frequency.The main differences in amino acid usege among the three groups were:The usege of methionine in SPF group was significantly higher than that in GF group(P<0.05).d.The diversity of memory B cells BCR H-CDR3 repertoire in small intestinal of three months old GF,SPF and CL BALB/cmice was different.The difference of 1/SD of memory B cell BCR H-CDR3 repertoire in small intestinal between GF group and SPF group was significant(P<0.05).The number of unique amino acid sequences shared by the three groups is 1.The first 10 high frequency cloned amino acid sequences in the three groups had highly conserved amino acid motifs "RYFDV" and"WYFDV",and GF group had the most "RYFDV" motif.The main differences between the three groups of nucleotide insertions and deletions are as follows:compared with GF group,the number of D3’ deleted nucleotides increased significantly in SPF group(P<0.05).There was no significant difference in the proportion of high frequency mutations of IGHV genes and the mutation rate of the mutation sequences in the three groups.(3)Sequencing and analysis results of memory B cells BCR H-CDR3 repertoire in spleen three months old GF,SPF and CL BALB/cmice(3 mice in each group)a.The number of unique sequences and total productive sequences of memory B cells BCR H-CDR3 repertoire in spleen of three months old GF,SPF and CL BALB/cmice(9 samples).MGF1-SP:2868/3144;MGF2-SP;173/185;MGF3-SP:688/733;MSPF1-SP:2044/2177;MSPF2-SP:1573/1684;MSPF3-SP:4078/4312;MCL1-SP:4932/5920;MCL2-SP:1454/1755;MCL3-SP:1410/1645;b.Memory B cells BCR H-CDR3 repertoire in spleen of three months old GF,SPF and CL BALB/cmice were all high frequency used IGHV09-03,IGHV03-02,IGHV06-06-06,IGHD01-01-01,IGHJ02-14,IGHJ04-01,IGHJ03-01.In comparison among the three groups,there was no significant difference in the gen usege of IGHD gene except for the gen usege of IGHV,IGHJ and V-J pairs.The main differences were as follows:The usege of IGHV05-06-2 in both SPF and CL groups was significantly lower than that in GF group(P<0.05).The usege of IGHJ02-01 in SPF group was significantly lower than that in GF group(P<0.05).Compared with GF group and SPF group,the gene pairing usege of IGHV05-06-3-IGHJ03-01 and IGHHV05-15-IGHJ03-01 and IGHV06-07-IGHJ03-01 in CL groups was significantly increased(P<0.05).c.The amino acids distribution of memory B cells BCR H-CDR3 in spleen of three months old GF,SPF and CL BALB/cmice were all bell-shaped with 11-12 amino acids as the midline and the hydrophobic amino acids represented by tyrosine were used at high frequency.The amino acids usege of memory B cells BCR H-CDR3 repertoire in spleen of three groups were different.The main differences were as follows:the usege of histidine and methionine in SPF and CL groups was significantly higher than that in GF group(P<0.05).d.The diversity index of memory B cell BCR H-CDR3 repertoire in spleen of GF,SPF and CL B ALB/cmice increased gradually,but there was no significant difference.The number of unique amino acid sequences shared by the three groups was 15.Three groups of the first 10 high frequency cloned amino acid sequences almost all have a highly conserved amino acid motif "WYFDV".The GF group also contains two conservative motifs"RYFDV" and "GYFDV".The main differences between the three groups of nucleotide insertions and deletions are as follows:compared with both other groups,the total number of nucleotides inserted at the broken end of CL group increased significantly(P<0.05).The main difference of the high frequency mutation sequence of IGHV gene among the three groups was that compared with the other two groups,the proportion of sequence with mutation rate less than 400 in CL group decreased significantly(P<0.05),but the proportion of sequence with mutation rate larger than 400 in CL group increased significantly(P<0.05).Conclusion:1.The colonization of intestinal flora may affect the development of the tissue structure of small intestine and lymph nodes,and the distribution of memory B cells in bone marrow,spleen and peripheral blood,and may also affect the immune response and tolerance of B cells.2.The usege frequency of pairing of V gene,J gene,and V-J gene pairs in BCR H-CDR3 repertoire of three-month-old GF-class,SPF-class,and CL-class BALB/c Mice with small intestinal memory B Cells,spleen memory B,and small intestine total B Cells and there were significant differences in the usege frequency of amino acid,and there were differences in the number of nucleotide insertions and deletions in the H-CDR3 repertoire.The diversity index(1/SD)of the BCR H-CDR3 repertoire of small intestine memory B cells of SPF grade BALB/c mice at 3 months of age was significantly higher than that of GF.Compared with GF and SPF grades,3 months old CL-class mice had a significantly higher proportion of IGHV gene mutations in their total small intestine B cells,and their IGHV gene mutation rate in splenic memory B cells was also significantly increased.A highly conserved motif in the BCR H-CDR3 repertoire of small intestine total B cells and intestinal memory B cells of 3-month-old GF-class BALB/c mice was the "WYFDV" and"RYFDV"motif,respectively. |
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