朝鲜鹌鹑MHC class Ⅰ基因第2、3外显子多态性与NDV抗性的关联分析
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摘要
【目的】研究朝鲜鹌鹑MHC classⅠ基因第1,2和3外显子的多态性,并与新城疫病毒抗性进行关联分析。【方法】采用胸肌注射的方法对14日龄朝鲜鹌鹑进行新城疫La Sota弱毒株攻毒,每周注射1次,连续攻毒3周后,心脏采血,采用β-微量法测定血清中新城疫抗体水平。PCR扩增朝鲜鹌鹑MHC classⅠ基因,测序后采用DNA Star、SPSS等分析软件研究朝鲜鹌鹑MHC classⅠ基因第1,2,3外显子的多态性,并分析不同基因型新城疫抗体水平的差异。【结果】朝鲜鹌鹑的新城疫抗体效价为26～210。PCR扩增获得了1 290bp的MHC classⅠ基因,其编码区序列为780bp,编码259个氨基酸。通过与已知序列日本鹌鹑RNA(AB005528)比对,得到长度分别为76,272和274bp的第1,2和3外显子。在第1外显子上未检测到突变为点,在第2和3外显子上共检测到11个突变位点(第2外显子上4个突变位点:257bp(A/C)、267bp(A/T)、366bp(A/G)和500bp(T/G);第3外显子上7个突变位点:795bp(T/C)、798bp(T/C)、810bp(T/G)、830bp(A/G)、873bp(A/T)、927bp(A/G)和932bp(C/G)),均为中度多态基因座,11个突变位点中,267bp(A/T)、830bp(A/G)、873bp(A/T)、932bp(C/G)突变导致编码氨基酸改变,500bp(T/G)、873bp(A/T)、932bp(C/G)突变位点基因型之间新城疫抗体水平存在显著差异(P<0.05)。【结论】朝鲜鹌鹑MHC classⅠ基因第2、3外显子具有丰富的多态性,并且与新城疫病毒抗性相关。
【Objective】The study analyzed the allelic polymorphism of exons 1,2 and 3 of MHC classⅠ gene in Korean quail and the association with the Newcastle disease virus(NDV)resistance.【Method】Based on conventional feeding method,fourteen-day-old Korean quails were challenged with La Sota attenuated strain of ND once a week by injection method.Three weeks after continuous challenge,blood samples were obtained from the heart.Theβ-micro method was used to determine the levels of NDV in serum.Amplification of MHCclass Ⅰgene in Korean quail by PCR technology,DNA Star and SPSS were used to analyze the allelic polymorphism of MHC class Ⅰ gene exons 2 and 3 and the NDV antibody differences among genotypes.【Result】The ND antibody valence in Korean quail was 26-210.PCR amplification obtained 1 290 bp DNA sequence and the coding region sequence was 780 bp,encoding 259 amino acids.DNA sequences of exons 1,2 and 3 of 76,272 and 274 bp were obtained by comparing with the known Japanese quail RNA(AB005528).No mutation was detected on exon 1,while 11 moderate polymorphic mutations loci were found in exons 2 and 3(4 mutations loci on exon 2:257 bp(A/C),267 bp(A/T),366 bp(A/G),and 500 bp(T/G);7 mutations loci on exon 3:795 bp(T/C),798 bp(T/C),810 bp(T/G),830 bp(A/G),873 bp(A/T),927 bp(A/G),and 932 bp(C/G).The 267 bp(A/T),830 bp(A/G),873 bp(A/T),and 932 bp(C/G)mutations led to amino acid changes.There were significant differences in ND antibody levels between the 500 bp(T/G),873 bp(A/T)and 932 bp(C/G)mutation loci(P<0.05).【Conclusion】The exons 2 and 3 of Korean quail MHC class Ⅰ gene had abundant polymorphism and close relationship with NDV resistance.
【Objective】The study analyzed the allelic polymorphism of exons 1,2 and 3 of MHC classⅠ gene in Korean quail and the association with the Newcastle disease virus(NDV)resistance.【Method】Based on conventional feeding method,fourteen-day-old Korean quails were challenged with La Sota attenuated strain of ND once a week by injection method.Three weeks after continuous challenge,blood samples were obtained from the heart.Theβ-micro method was used to determine the levels of NDV in serum.Amplification of MHCclass Ⅰgene in Korean quail by PCR technology,DNA Star and SPSS were used to analyze the allelic polymorphism of MHC class Ⅰ gene exons 2 and 3 and the NDV antibody differences among genotypes.【Result】The ND antibody valence in Korean quail was 26-210.PCR amplification obtained 1 290 bp DNA sequence and the coding region sequence was 780 bp,encoding 259 amino acids.DNA sequences of exons 1,2 and 3 of 76,272 and 274 bp were obtained by comparing with the known Japanese quail RNA(AB005528).No mutation was detected on exon 1,while 11 moderate polymorphic mutations loci were found in exons 2 and 3(4 mutations loci on exon 2:257 bp(A/C),267 bp(A/T),366 bp(A/G),and 500 bp(T/G);7 mutations loci on exon 3:795 bp(T/C),798 bp(T/C),810 bp(T/G),830 bp(A/G),873 bp(A/T),927 bp(A/G),and 932 bp(C/G).The 267 bp(A/T),830 bp(A/G),873 bp(A/T),and 932 bp(C/G)mutations led to amino acid changes.There were significant differences in ND antibody levels between the 500 bp(T/G),873 bp(A/T)and 932 bp(C/G)mutation loci(P<0.05).【Conclusion】The exons 2 and 3 of Korean quail MHC class Ⅰ gene had abundant polymorphism and close relationship with NDV resistance.
引文
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[24]Paul S,Weiskopf D,Angelo M A,et al.HLA classⅠalleles are associated with peptide binding repertoires of different size,affinity and immunogenicity[J].Journal of Immunology,2013,191(12):5831-5839.
[25]Bolton L,Bailey A,Findlow S,et al.Structural plasticity and antigen selection by MHCⅠ[J].Molecular Immunology,2015,68(2):143-145.
[26]Liu W,Miller M M,Lamont S J.Association of MHC classⅠand classⅡgene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks[J].Immunogenetics,2002,54:582-590.
[2]Grubic Z,Jankovic K S,Maskalan M,et al.The effect of HLAallele and haplotype polymorphisms on donor matching in hematopoietic stern cell transplantation croatian experience[J].Human Immunology,2016,77(12):1120-1127.
[3]Morita D,Yamamoto Y,Mizutani T,et al.Crystal structure of the N-myristoylated lipopetide-bound MHC classⅠcomplex[J].Nature Communications,2016,7:10356-10367.
[4]Backer M.Accessory proteins and the assembly of human classⅠMHC molecules:a molecular and structural perspective[J].Molecular Immunology,2003,39:697-706.
[5]Carlini F,Ferreira V,Buhler S,et al.Association of HLA-Aand non-classical HLA classⅠalleles[J].PloS One,2016,11(10):e0163570.
[6]Biedrzycka A,Kloch A.Development of novel associations between MHC alleles and susceptibility to parasitic infections in an isolated population of an endangered mammal[J].Infection Genetics and Evolution,2016,44:210-217.
[7]Micheli A.A rare constellation of serum cholinesterase genetic variants[J].Enzyme,1972,13(5/6):305-310.
[8]van H A,Carter R,Bailey A,et al.A mechanistic basis for the co-evolution of chicken tapasin and major histocompatibility complex classⅠ(MHCⅠ)proteins[J].Journal of Biological Chemistry,2013,288(45):32797-32808.
[9]Bauer M M,Miller M M,Briles W E,et al.Genetic variation at the MHC in a population of introduced wild turkeys[J].Animal Biotechnology,2013,24(3):210-228.
[10]Shiina T,Briles W E,Hosomichi K,et al.Extended gene map reveals tripartite motif,C-type lectin,and Ig superfamily type genes within a subregion of the chicken MHC-B affecting infectious disease[J].Journal of Immunology,2007,178(1):7162-7172.
[11]Delany M E,Robinson C M,Goto R M,et al.Architecture and organization of chicken micro chromosome 16:order of the NOR,MHC-Y,and MHC-B subregions[J].Journal of Heredity,2009,100(5):507-514.
[12]Wang Y,Qiu M,Yang J,et al.Sequence variations of the MHCclassⅠ,gene exon 2and exon 3between infected and uninfected chickens challenged with Marek’s disease virus[J].Infection Genetics and Evolution,2014,21:103-109.
[13]姚火春.兽医微生物学实验指导[M].2版.北京:中国农业出版社,2002.Yao H C.Veterinary microbiology experimental guidance[M].2nd ed.Beijing:China Agricultural Press,2002.
[14]雷雪芹.牛羊多态性状的四种分子遗传标记研究[D].陕西杨凌:西北农林科技大学,2005.Lei X Q.Study on four molecular genetic markers of polymorphic traits in cattle and sheep[D].Yangling,Shaanxi:Northwest A&F University,2005.
[15]甘乾福.福建黄牛地方品种遗传多样性研究[D].福州:福建农林大学,2006.Gan Q F.Study on genetic diversity of local breed of Fujian Yellow Cattle[D].Fuzhou:Fujian Agriculture And Forestry University,2006.
[16]Deist M S,Gallardo R A,Bunn D A,et al.Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome[J].Bmc Genomics,2017,18(1):989-1004.
[17]Das M,Kumar S.Recombinant phosphoprotein based single serum dilution ELISA for rapid serological detection of Newcastle disease virus[J].Journal of Virological Methods,2015,225:64-69.
[18]Zhou H,Lamont S J.Chicken MHC classⅠandⅡgene offects on antibody response kinetics in adult chickens[J].Immunogenetics,2003,55(3):133-140.
[19]Xia C,Lin Y,Hu T J,et al.cDNA cloning and genomic structure of the duck(Anas platyrhynchos)MHC classⅠgene[J].Immunogenetics,2004,56:306-309.
[20]Xia C,Hu T J,Yang T Y,et al.cDNA cloning,genomic structure and expression analysis of the goose(Anser cygnoides)MHC classⅠgene[J].Veterinary Immunology and Immunopathology,2005,107(3/4):291-302.
[21]Altmann D M,Hogg N,Trowsdale J,et al.Cotransfection of ICAM-1and HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells[J].Nature,1999,338(6215):512-514.
[22]Fremont D H,Matsumura M,Stura E A,et al.Crystal structures of two viral peptides in complex with murine MHC classⅠH-2Kb[J].Science,1992,257(5072):919-927.
[23]Madden D R,Garboczi D N,Wiley D C.The antigenic identity of peptide-MHC complexes:a comparison of the conformations of five viral peptides presented by HLA-A2[J].Cell,1993,75(4):693-708.
[24]Paul S,Weiskopf D,Angelo M A,et al.HLA classⅠalleles are associated with peptide binding repertoires of different size,affinity and immunogenicity[J].Journal of Immunology,2013,191(12):5831-5839.
[25]Bolton L,Bailey A,Findlow S,et al.Structural plasticity and antigen selection by MHCⅠ[J].Molecular Immunology,2015,68(2):143-145.
[26]Liu W,Miller M M,Lamont S J.Association of MHC classⅠand classⅡgene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks[J].Immunogenetics,2002,54:582-590.