| The individualized medication of treatment based on syndrome differentiation is the eternal concept of traditional Chinese medicine,while,the development of genomics has provided theoretical support for individualized use of drugs.The compound Chinese herbal medicine polysaccharides,a kind of T cell-dependent antigen,can be processed and presented by MHC class II molecules,and it can be recognized and combined with TCR to participate in immune activity.Due to the high polymorphism of MHC B-L?? gene in different individuals,MHC class II epitope binding groove has a certain degree of selectivity for the acceptance and presentation of exogenous epitopes and the induction of specific immunity to T and B cells.Therefore,different individuals have different immune responses to external antigens,which may affect the selection of optimal immunomodulating doses of polysaccharides.Object:In this study,different MHC B-L beta II egg laying chicks were used to explore the immunoregulation effect of the compound Chinese herbal medicine polysaccharides(purity 77.1%),which provided a theoretical basis for the development of the compound Chinese herbal medicine polysaccharides as an immune enhancer.Methods:1.PCR-SSCP technique was used to screen the different genotypes of the exon 3 of MHC B-L?? gene in 500 hens of Jinghong 1.The test chickens were grouped according to their genotypes and the same genotype chickens were subdivided into four groups,then,were intramuscular injection the compound Chinese herbal medicine polysaccharides with 50 mg/mL(high dose),25 mg/m L(medium dose),12.5mg/m L(low dose)and normal saline(control group)at 8 days of age,respectively.The following items were examined at 7 days post-injection.2.The thymus,spleen and bursa of chicks were collected at 21,35 and 49 days of age to calculate the index of immune organs.The blood routine of peripheral blood of 21,27,35,42 and 49 day-old chicks was detected by animal blood analyzer.At the age of 28 days,1 mL sheep erythrocyte solution was injected intramuscularly into leg of the each chick,and the SRBC antibody titers in the serum of 35,42,and 49 days old chicks were detected by the hemagglutination test.3.The serum concentrations of IL-1?,IL-10,IL-15,G-CSF,and GM-CSF and antibody titer of IBDV were detected by ELISA in 21,27,35,42,and 49 days old chicks.And,hemagglutination inhibition test(HI)was used to detect the titers of H5 and H9 subtype AI-HI antibodies in serum of 27,35,42,and 49 days old chicks.Results:1.According to PCR-SSCP results,three genotypes were found,which included AA(103 feathers),BB(266 feathers)and BC(131 feathers).The results of gene sequencing analysis showed that the AA genotype was identical to the sequence published in Genbank and had no mutation sites;the BB genotype had a mutation site(G1722A),and its corresponding to the encoded amino acid changed from Val to Met;the BC genotype had two mutation sites(T1597A and G1722A),and its corresponding encoded amino acids changed from Leu to Gln and Val to Met,respectively.2.Compared with the control group,high-dose group can significantly increase the bursa index of AA genotypic chicks(P<0.05),middle-dose group can significantly improve the thymus index of AA genotype chicken(P<0.05),middle-and high-dose group can significantly increase the index of thymus,spleen,and bursa viscera in the pre-test period of BC genotype chicks and significantly increase the total number of leukocytes and lymphocytes of AA and BC genotype chicks(P<0.05).One week after intramuscular injection sheep red blood cells,the SRBC titers in the test groups were 0.6-1.36 titers higher than that in the control group.3.Compared with the control group,the test groups can significantly increase the concentration of IL-1?,IL-15,GM-CSF and reduce the concentration of IL-10 in serum,while the G-CSF quality concentration was not significant with control.At the same time,the compound Chinese herbal medicine polysaccharides had different effect on the cytokine concentration of different genotype chicks.Compared with other dose groups,the middle dose group can significantly increase the serum concentrations of IL-1?and IL-15 and significantly reduce the concentration of IL-10 in AA genotype chicks(P<0.05);the high dose group can significantly increase the serum concentration of IL-1?,IL-15 in BB genotype chicks(P<0.05).There was no significant difference among the experimental groups in BC genotype chicks.In addition,the compound Chinese herbal medicine polysaccharides group can significantly increase the antibody titer of IBDV antibody in chicken serum(P<0.05),and the best effect dose was the middle dose for AA genotype chicks,high-dose group for BB genotype chicks,however,there was no significant difference between BC genotype chicks.Compared with the control group,the compound Chinese herbal medicine polysaccharides groups had a certain maintenance effect on maternal H5,H9 subtype AI antibody titers.After immunization the H5 and H9 double vaccines,the titer of H5 and H9 subtype AI antibody of each genotype chicks in the high-and medium-dose groups was 0.4-1.6 titer higher than that of the control group,and there was a tendency for the BC genotype chicks to prolong the antibody level of H5 and H9 subtypes AI antibody titers.Conclusion:1.Three genotypes of AA(103 feathers),BB(266 feathers)and BC(131 feathers)in exon 3 of the MHC B-L?? gene,of which the BB genotype had a G1722 A mutation site,and the BC genotype had two mutation sites T1597 A and G1722 A.Both mutation sites caused mutations in the corresponding coding amino acids and were effective mutations.2.After using the compound Chinese herbal medicine polysaccharides,the index of immune organs,the number of leukocyte,and SRBC antibody titer of different genotypes showed different degrees ofimprovement.It was initially judged that the immune enhancing effect of compound Chinese herbal medicine polysaccharides on different MHC B-L?? genotype chicks was different,and the presence of genetic polymorphisms may have affected the selection of traditional Chinese medicine compound polysaccharide doses.3.After using compound Chinese herbal medicine polysaccharides,the concentration of IL-1? and IL-15 and the antibody titer of IBDV and H5,H9 subtype AI vaccines in serum of different genotype chicks were significantly increased,and the concentration of IL-10 was significantly reduced.While the most effective doses of polysaccharides in different genotype chicks were different in regulating the concentration of relevant cytokines and the antibody titer of the vaccines,which indicated again that the presence of genetic polymorphisms may affect the screening of polysaccharide doses of traditional Chinese medicine compound. |
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