Studies on the Physiological Function of Lymphocytes and the Polymorphism of Major Histocompatibility Complex in Japanese Quails

日本鹌鹑淋巴细胞生理功能及主要组织相容性复合物多态性的研究

• 批准号: 04806040
• 负责人: WATANABE Seiki
• 金额: $ 1.28万
• 依托单位: Tokyo University of Agriculture
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04806040/
• 关键词: Japanese quail; Lymphocyte; T cell; B cell; Monoclonal antibody; MHC class I; RFLPs; cDNA; MHCクラスI; 血清IgG値; モノクロール抗体; マイトージェン; 抗体産性能; MHC; ロゼット形成細胞; ロゼット非形成細胞

1.Studies on the lymphocyte antigens of Japanese quail : (1) As hybridomer of 5 clones derived from thymocyte : QT-1-QT-5 ; and 5 clones derived from bursacyte : QB-1-QB-5.(2) Imminohistochemical analysis on the immuno organ were carried out by the monoclonal anitibodies. QT-1^+, -4^+ and 5^+ cells only exist incortex of thymus.QB-1^+, -2^+ and -3^+ cells exist in medulla or bursa of Fabricius and in B cell locus of PALS of spleen.(3) Lymphocyte antigens were analyzed by flowcytometory. QT-1^+, -4^+ and 5^+ cells only exist thymus and their frequencies were 70 - 80%. The frequencies of QB-1^+, -2^+ and -3^+ cells were 90% in brusacytes.2.Analysis of major histocompatibility complex (MHC) class I gene from Japanese quail : (1) RFLPs patten of genomic DNA from Japanese quail which were analyzed using BF10 in chicken MHC class I cDNA as a probe showed polymorphism.(2) Nine positive clones : QF41,63,76,95,103,111,113,128 ; corresponding to the MHC class I gene of Japanese quail were isolated by screening of liver cDNA library with BF10 and QF41 as arobe. Based on comparison among sequences of QF clones, MHC class I of Japanese quail classified into 5 loci.(3) PCR analysis of alpha1-alpha2 region using genomic DNA as a template with QF63 and QF41 specific primers were revealed different sizes of fragment by 609bp in PCR product of QF63 vs 434bp in QF41 product. QF63 has 181bp intron between alpha1 and alpha2 regions, but QF41 have not intron between alpha1 and alpha2 regions.(4) PCR products using the QF63 specific primer were digested with the 2 kinds of restriction endnuclease : Ban II and EcoT14 I.Combination of PCR-RFLP patterns using 2 kinds of restriction endnuclease classified 9 haplotypes QF63 product, but only 5 haplotypes ovserved in this experiment.

1.日本鹌鹑淋巴细胞抗原的研究：（1）以胸腺细胞来源的5个克隆：QT-1-QT-5和囊细胞来源的5个克隆：QB-1-QB-5作为杂交瘤。(2)用单克隆抗体对免疫器官进行免疫组织化学分析。QT-1^+、-4 ^+和5^+细胞仅存在于胸腺结合部，Q B-1^+、-2 ^+和-3 ^+细胞存在于腔上囊或髓质以及脾脏帕尔斯的B细胞部位。(3)流式细胞术分析淋巴细胞抗原。QT-1^+、-4 ^+和5^+细胞仅存在于胸腺中，其频率为70 - 80%。2.日本鹌鹑主要组织相容性复合体（MHC）I类基因的分析：（1）以鸡MHC I类基因cDNA中的BF 10为探针，对日本鹌鹑基因组DNA进行RFLP分析，结果表明日本鹌鹑基因组DNA具有多态性。(2)9个阳性克隆：以BF 10和QF 41为载体，从日本鹌鹑肝脏cDNA文库中筛选到与日本鹌鹑MHC Ⅰ类基因相对应的QF 41、63、76、95、103、111、113、128。根据QF克隆序列的比较，日本鹌鹑MHC Ⅰ类分子可分为5个位点。(3)以QF 63和QF 41的基因组DNA为模板，对α 1-α 2区进行PCR分析，结果表明，QF 63的PCR产物中有609 bp的片段，而QF 41的PCR产物中有434 bp的片段。QF 63在α 1和α 2区之间有181 bp的内含子，而QF 41在α 1和α 2区之间没有内含子。(4)用QF 63特异性引物PCR产物经Ban Ⅱ和EcoT 14 Ⅰ两种限制性内切酶酶切，结合PCR-RFLP图谱，将QF 63产物分为9种单倍型，而本实验只观察到5种单倍型。