小鼠H-2基因位点遗传检测技术的研究

发布时间：2018-05-30 13:56

本文选题：近交系小鼠 + 遗传检测　；参考：《中国药品生物制品检定所》2005年硕士论文

【摘要】：H-2基因复合体是决定小鼠免疫遗传品质最主要的基因群，其中以K、D基因区段产物的免疫反应性最强，最具决定性。因此建立小鼠H-2检测技术，可保障和控制实验小鼠的免疫遗传质量，完善我国实验动物遗传检测技术。本研究分三个部分。首先建立了微量细胞毒法，引进小鼠主要组织相容性复合体国际标准单克隆抗体系列—H-2D~b(27-11-13)、H-2D~d(34-5-8)、H-2D~k(15-5-5.3)、H-2K~k(16-3.22.4)，和国产单克隆抗体H-2D~b(2)、H-2D~d(4)、H-2D~k(32)、H-2K~b(33)、H-2K~d(31)、H-2K~k(23)。制取小鼠脾细胞，加入特异的单克隆抗体，结果显示抗原与特异抗体发生强烈的免疫反应，导致同型的脾细胞死亡，通过倒置显微镜观察死亡细胞占总细胞的百分比来判定不同品系小鼠H-2基因型。同时进行了国内外单克隆抗体的对比研究，结果表明两种抗体没有显著性差别。实验的第二部分，根据H-2D区和H-2K区的DNA序列，利用Megalin程序进行多序列比较，在各品系小鼠之间相同的区域内设计引物，扩增各品系小鼠之间不同的序列。H-2D区扩增片段为828bp，H-2K区扩增片段为104bp。经过纯化、连接、转化、测序验证扩增结果的准确性后，通过PCR方法扩增出特异区，从聚丙烯酰胺凝胶电泳图谱中可观察到不同品系的小鼠H-2基因型表现出不同的电泳条带，从而可以区分不同的品系。实验的第三部分，为了提高实验的灵敏度和特异度，，建立了微孔
[Abstract]:H-2 gene complex is the most important gene group which determines the immune genetic quality of mice. Therefore, the establishment of mouse H-2 detection technology can guarantee and control the immunogenetic quality of experimental mice and improve the genetic detection technology of laboratory animals in China. This study is divided into three parts. In this paper, the method of microcytotoxicity was first established, and the international standard monoclonal antibody series of major histocompatibility complex of mice, -H-2DBU 27-11-13C, H-2Ddc34-5-5-8, H-2DOKCU, 15-5-5-5.3, H-2DGK, 16-3.22.4H, and the domestic monoclonal antibody, H-2Db2Dd4, H-2Ddt4, were introduced. Mouse spleen cells were prepared and specific monoclonal antibodies were added. The results showed that the antigen reacted strongly with the specific antibodies, resulting in the death of the same type of spleen cells. The percentage of dead cells in total cells was observed by inverted microscope to determine the H-2 genotypes of different strains of mice. The results showed that there was no significant difference between the two antibodies. In the second part of the experiment, according to the DNA sequence of H-2D region and H-2K region, the multiple sequences were compared by Megalin program, and primers were designed in the same region among all strains of mice. The sequence of different strains of mice was amplified from 828bpnH-2K to 104bp2.The results showed that the amplified fragment of H-2D region was 828bp2D and the amplified fragment was 104bp2K. After purification, ligation, transformation and sequencing to verify the accuracy of the amplification results, the specific regions were amplified by PCR method. Different bands of H-2 genotypes of different strains of mice were observed from polyacrylamide gel electrophoresis atlas. Thus, different strains can be distinguished. In the third part of the experiment, in order to improve the sensitivity and specificity of the experiment, the micropore was established.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R-332

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