發(fā)布時(shí)間：2018-04-22 03:22

  本文選題：人心肌肌鈣蛋白I + 單克隆抗體��； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 肌鈣蛋白復(fù)合物由肌鈣蛋白C、肌鈣蛋白I和肌鈣蛋白T三個(gè)不同的亞基組成,在Ca~(2+)作用下,主要參與肌肉的收縮調(diào)節(jié)。肌鈣蛋白I作為一個(gè)亞單位主要抑制肌球蛋白ATP酶的活性。心肌肌鈣蛋白I不在任何骨骼肌組織中表達(dá),具有心肌組織特異性,這使得其成為檢測(cè)心肌損傷最好的生化標(biāo)志物之一。研究表明急性心肌梗死患者胸痛發(fā)生的早期心肌肌鈣蛋白I就被釋放入血流,14～36小時(shí)其濃度達(dá)到最高峰。心肌損傷后的3～7天仍能檢測(cè)到心肌肌鈣蛋白I水平的升高。因此,心肌損傷的檢測(cè)可通過(guò)檢測(cè)血清中心肌肌鈣蛋白I的水平來(lái)進(jìn)行,并且具有靈敏度高、特異性強(qiáng)和診斷窗口期長(zhǎng)的特點(diǎn)。進(jìn)一步研究表明急性冠脈綜合癥死亡風(fēng)險(xiǎn)與心肌肌鈣蛋白I的水平具有相關(guān)性。在一些非致死的心肌損傷、充血性心衰等心臟疾病中也檢測(cè)到心肌肌鈣蛋白I水平的升高。目前,心肌肌鈣蛋白I已成為臨床上診斷心肌損傷的“金標(biāo)準(zhǔn)”。 本研究的目的是制備重組人心肌肌鈣蛋白I(recombinant human cardiactroponinI,rhcTnI)的單克隆抗體,并對(duì)其特性進(jìn)行鑒定,為cTnI診斷試劑盒的研制奠定基礎(chǔ)。 利用已構(gòu)建的基因工程菌株,大量表達(dá)、純化重組目的蛋白。純化后的目的蛋白作為抗原常規(guī)免疫Balb/c小鼠,取其脾細(xì)胞與同系骨髓瘤細(xì)胞Sp2/0進(jìn)行常規(guī)融合,甲基纖維素半固體培養(yǎng)基法篩選雜交瘤細(xì)胞株,利用ELISA、Western blotting等方法鑒定單克隆抗體的性質(zhì)。 篩選出3株穩(wěn)定分泌抗重組目的蛋白抗體的雜交瘤細(xì)胞株,分別命名為4B_3、7D_5和3E_6,所產(chǎn)生單克隆抗體的免疫球蛋白類及亞類分別為IgGl(48_3、7D_5)和IgG2b(3E_6)。單克隆抗體與重組目的蛋白發(fā)生特異性結(jié)合,而與空白菌、LDH、CK、CK-MB無(wú)特異性交叉反應(yīng),單克隆抗體的親和常數(shù)分別為1.76×10~9L/mol、1.43×10~9L/mol和1.04×10~9L/mol。 綜上所述,成功獲得了親和力高、特異性好的單克隆抗體。
[Abstract]:The troponin complex is composed of three different subunits, troponin C, troponin I and troponin T. It is mainly involved in the contractile regulation of muscle under the action of Ca~ (2+). The troponin I as a subunit mainly inhibits the activity of myosin ATP enzyme. Cardiac troponin I is not expressed in any skeletal muscle tissue and has myocardial tissue. Specificity, which makes it one of the best biochemical markers for detecting myocardial damage, has shown that early cardiac troponin I in acute myocardial infarction is released into the blood flow and its concentration reaches its peak at 14~36 hours. The cardiac troponin I level can still be detected at the 3~7 day after myocardial injury. The detection of muscle damage can be carried out by detecting the level of cardiac troponin I in serum, with high sensitivity, specificity, and long period of diagnosis. Further studies show that the risk of acute coronary syndrome is associated with the level of cardiac troponin I. In some nonfatal myocardial injuries, congestive heart failure Cardiac troponin I levels have also been detected in heart diseases. At present, cardiac troponin I has become the "gold standard" for clinical diagnosis of myocardial injury.
The purpose of this study is to prepare monoclonal antibodies against recombinant human cardiac troponin I (recombinant human cardiactroponinI (rhcTnI)), and to identify the properties of the recombinant human cardiac troponin cardiactroponinI (rhcTnI), which lays the foundation for the development of cTnI diagnostic kit.
Using the constructed genetic engineering strain to express and purify the recombinant target protein, the purified target protein was used as the antigen routine immunization Balb/c mice, and the spleen cells were fused with the homologous myeloma cell Sp2/0, the methylation semisolid culture method was used to screen the hybridoma cell lines, and the methods of ELISA, Western blotting and other methods were used. Identify the properties of the monoclonal antibody.
3 hybridoma cell lines that secrete anti recombinant target protein antibodies were selected, named 4B_3,7D_5 and 3E_6 respectively. The immunoglobulins and subclasses of monoclonal antibodies were IgGl (48_3,7D_5) and IgG2b (3E_6) respectively. The monoclonal antibody combined with the recombinant target protein, and was not specific to the blank bacteria, LDH, CK and CK-MB. The affinity constants of the monoclonal antibodies were 1.76 x 10~9L/mol, 1.43 x 10~9L/mol and 1.04 x 10~9L/mol., respectively.
In conclusion, the monoclonal antibody with high affinity and specificity has been successfully obtained.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 萬(wàn)文徽;單克隆抗體親和常數(shù)的測(cè)定[J];細(xì)胞與分子免疫學(xué)雜志;1993年02期

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本文編號(hào)：1785382