發(fā)布時(shí)間：2018-05-30 13:56

  本文選題：近交系小鼠 + 遺傳檢測(cè)　； 參考：《中國(guó)藥品生物制品檢定所》2005年碩士論文

【摘要】：H-2基因復(fù)合體是決定小鼠免疫遺傳品質(zhì)最主要的基因群，其中以K、D基因區(qū)段產(chǎn)物的免疫反應(yīng)性最強(qiáng)，最具決定性。因此建立小鼠H-2檢測(cè)技術(shù)，可保障和控制實(shí)驗(yàn)小鼠的免疫遺傳質(zhì)量，完善我國(guó)實(shí)驗(yàn)動(dòng)物遺傳檢測(cè)技術(shù)。 本研究分三個(gè)部分。首先建立了微量細(xì)胞毒法，引進(jìn)小鼠主要組織相容性復(fù)合體國(guó)際標(biāo)準(zhǔn)單克隆抗體系列—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)，和國(guó)產(chǎn)單克隆抗體H-2D~b(2)、H-2D~d(4)、H-2D~k(32)、H-2K~b(33)、H-2K~d(31)、H-2K~k(23)。制取小鼠脾細(xì)胞，加入特異的單克隆抗體，結(jié)果顯示抗原與特異抗體發(fā)生強(qiáng)烈的免疫反應(yīng)，導(dǎo)致同型的脾細(xì)胞死亡，通過(guò)倒置顯微鏡觀察死亡細(xì)胞占總細(xì)胞的百分比來(lái)判定不同品系小鼠H-2基因型。同時(shí)進(jìn)行了國(guó)內(nèi)外單克隆抗體的對(duì)比研究，結(jié)果表明兩種抗體沒(méi)有顯著性差別。 實(shí)驗(yàn)的第二部分，根據(jù)H-2D區(qū)和H-2K區(qū)的DNA序列，利用Megalin程序進(jìn)行多序列比較，在各品系小鼠之間相同的區(qū)域內(nèi)設(shè)計(jì)引物，擴(kuò)增各品系小鼠之間不同的序列。H-2D區(qū)擴(kuò)增片段為828bp，H-2K區(qū)擴(kuò)增片段為104bp。經(jīng)過(guò)純化、連接、轉(zhuǎn)化、測(cè)序驗(yàn)證擴(kuò)增結(jié)果的準(zhǔn)確性后，通過(guò)PCR方法擴(kuò)增出特異區(qū)，從聚丙烯酰胺凝膠電泳圖譜中可觀察到不同品系的小鼠H-2基因型表現(xiàn)出不同的電泳條帶，從而可以區(qū)分不同的品系。 實(shí)驗(yàn)的第三部分，為了提高實(shí)驗(yàn)的靈敏度和特異度，，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.
【學(xué)位授予單位】：中國(guó)藥品生物制品檢定所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王莉,吳玉章,林治華,石統(tǒng)東,周偉,賈正才,鄒麗云;穿膜序列對(duì)MHC-Ⅰ類限制性CTL表位呈遞影響的研究[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2002年10期

2 李善如,葛寶生,陳永福,張忠誠(chéng);遺傳標(biāo)記及其在動(dòng)物遺傳育種中的應(yīng)用[J];國(guó)外畜牧科技;1997年01期

3 龔浩,李龍江,溫玉明,王昌美,陳俊杰,彭文珍;615小鼠MHC Ⅰ cDNA重組逆轉(zhuǎn)錄病毒基因治療載體的構(gòu)建和包裝[J];華西口腔醫(yī)學(xué)雜志;2002年06期

4 金玫蕾,張瑞忠,鮑世民,趙國(guó)際;小鼠H-2,Hc和Thy-1單價(jià)特異抗血清的制作研究[J];免疫學(xué)雜志;1991年04期

5 孫瑋,葛海良,王穎,張惠珍,張笑人,周光炎;轉(zhuǎn)染MHCII類基因?qū)β殉舶┘?xì)胞株免疫特性的影響[J];上海免疫學(xué)雜志;2003年03期

6 張瑞忠,鮑世民,金玫蕾;微量細(xì)胞毒檢測(cè)技術(shù)及其在實(shí)驗(yàn)小鼠遺傳監(jiān)測(cè)中的應(yīng)用[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);1998年Z1期

7 ;《上海實(shí)驗(yàn)動(dòng)物科學(xué)》第24卷 第1~4期總目次[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2004年04期

8 張建平,朱勇,張婷婷,孫凱,劉飛,金伯泉;H-2~b和H-2~d單體型小鼠MHCI類分子結(jié)合的HFRSV蛋白抗原表位的預(yù)測(cè)[J];細(xì)胞與分子免疫學(xué)雜志;1998年04期

9 邢瑞昌,宋珍珠,劉雙環(huán),李秀華,胡宗漢,李德富;用于乙肝疫苗效力檢定的NIH-q小鼠種群的建立[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)雜志;1991年01期

10 劉雙環(huán)，邢瑞昌;實(shí)驗(yàn)小鼠遺傳檢測(cè)試劑盒的研制及應(yīng)用[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)雜志;1996年03期

本文編號(hào)：1955540