本文關(guān)鍵詞：肝臟F蛋白的表達(dá)及單克隆抗體的制備　出處：《天津醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： F蛋白 肝臟 抗體 單克隆 免疫

【摘要】： 肝臟疾病嚴(yán)重影響人類健康,并對(duì)社會(huì)經(jīng)濟(jì)發(fā)展產(chǎn)生巨大影響。肝癌是世界上死亡率最高的惡性腫瘤之一,病毒性肝炎、肝纖維化、肝硬化等都是肝癌主要的致病原。因此肝病的診斷就成為醫(yī)學(xué)診斷學(xué)的重要組成部分。人類肝臟F蛋白是一種新型的尚未應(yīng)用于臨床的直接反映肝損傷程度的生物學(xué)標(biāo)志之一。從80年代開始,人們開始研究血清F蛋白含量與肝損傷程度的關(guān)系,獲得了一些臨床數(shù)據(jù)。肝細(xì)胞中的F蛋白濃度與血清F蛋白的濃度之間存在的巨大差異以及F蛋白的嚴(yán)格的組織特異性,使其成為一種非常有希望的反映肝細(xì)胞損傷程度的靈敏和特異的指標(biāo)。目前,肝臟特異性F蛋白的研究與臨床應(yīng)用受到越來越多的關(guān)注。 本文在前人研究的基礎(chǔ)上,將F蛋白的表達(dá)質(zhì)粒pET15b-F轉(zhuǎn)化到表達(dá)菌株BL21(DE3)pLysS中,用IPTG誘導(dǎo)其表達(dá),表達(dá)后通過親和層析柱(Ni~(2+)-chargedIDA His-bind column)純化,用十二烷基硫酸鈉-聚丙稀酰胺凝膠電泳與免疫印跡(Western blot)對(duì)其進(jìn)行檢測(cè)。并以此蛋白為抗原免疫BALB-C小鼠以獲得免疫的脾細(xì)胞,用間接ELISA法測(cè)定其血清抗體的效價(jià),選取抗體效價(jià)高的小鼠取脾細(xì)胞與骨髓瘤細(xì)胞融合,制備單克隆抗體。 人F蛋白的表達(dá)菌株BL21(DE3)pLysS表達(dá)后的全菌蛋白進(jìn)行SDS-PAGE電泳檢測(cè),目的條帶分子量大小約為43kD,與文獻(xiàn)報(bào)導(dǎo)一致。Western-blot顯示純化后的F蛋白與豚鼠抗人提純F蛋白多克隆抗血清反應(yīng)為陽性,說明我們經(jīng)基因工程方法得到的純化的F蛋白具有免疫活性。免疫BALB-C小鼠后,用ELISA法測(cè)定血清其抗體滴度高達(dá)1∶72000,可用于單抗的制備;細(xì)胞融合后培養(yǎng)上清有15孔抗體陽性,抗體滴度最高為1:25600,Western-blot顯示制備的抗體與基因工程表達(dá)的人肝臟特異性F蛋白發(fā)生了反應(yīng),表明我們制備的抗體具有免疫特異性,可用于后續(xù)的單克隆抗體的大量制備和提純以及免疫試劑的開發(fā)。 肝臟特異性F蛋白是反映肝損傷的一種靈敏、有效而又穩(wěn)定的指標(biāo),同時(shí)F蛋白在肝病早期診斷與治療、藥物的篩選與療效觀察、治愈狀況、肝損傷程度的判斷等方面以及法醫(yī)學(xué)方面、免疫耐受肝移植抗排斥方面的研究都具有重要的應(yīng)用價(jià)值。用化學(xué)提純的方法很難得到大量F蛋白,這就阻礙了其他研究的進(jìn)行。因此,本文采用基因工程方法得到人肝臟F蛋白的表達(dá)克隆,并誘導(dǎo)表達(dá)人肝臟F蛋白,解決了其來源問題。同時(shí)以此基因工程蛋白為抗原初步制備單克隆抗體,并通過免疫印跡法證實(shí)了此抗體的特異性,為下一步的免疫試劑最適條件的摸索、免疫耐受及其免疫學(xué)特性等研究打下基礎(chǔ),為臨床肝病的診斷和治療打開新的一扇門。
[Abstract]:Liver diseases seriously affect human health, and have a huge impact on the social and economic development. Liver cancer is one of malignant tumors with the highest mortality rate in the world, viral hepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma are the main pathogens. Therefore the diagnosis of liver disease has become an important part of medical diagnostics. The F protein in human liver is a new kind of biological mark is not applied to clinical directly reflect the degree of liver injury. From the beginning of 80s, people began to study the relationship between serum F protein content and the degree of liver injury, get some clinical data. There is a huge difference between the concentration of F protein in liver cells and the serum concentration of F protein and F protein the strict tissue specificity, make it become a very promising to reflect the degree of liver cell injury of the sensitive and specific index. At present, the research of the liver specific F protein More and more attention has been paid to the clinical application.
In this paper, on the basis of previous studies, the expression of transforming plasmid pET15b-F F protein expression strain BL21 (DE3) pLysS, induced the expression of IPTG expression by affinity chromatography (Ni~ (2+) -chargedIDA His-bind column) with twelve purification, sodium dodecyl sulfate - poly acrylamide gel electrophoresis and Western blot (Western blot) was used to detect the protein. Then BALB-C mice were immunized with immune spleen cells, the serum antibody titer was determined by indirect ELISA method, selection of high antibody titer of mice spleen cells and myeloma cell fusion, preparation of monoclonal antibody.
Human F protein expression strain BL21 (DE3) and the protein expression of pLysS was detected by SDS-PAGE, the target band size of molecular weight is about 43kD, and the purified F protein and F protein purified guinea pig anti human polyclonal antibody response was positive reported consistent.Western-blot showed that we by gene engineering method the purified F protein with immunological activity. After immunization of BALB-C mice, the ELISA method for the determination of serum antibody titers were as high as 1 to 72000, can be used for the preparation of the monoclonal antibody; cell culture supernatant after fusion of 15 hole antibody positive and antibody titer as high as 1:25600, Western-blot shows the reaction expression of antibody and gene engineering the preparation of human liver specific F protein showed that the antibodies we prepared has immune specificity, can be used for the large number of monoclonal antibody preparation and purification and immunological reagents.
The liver specific F protein is a reflection of liver injury in a sensitive, effective and stable index, and F protein in the early diagnosis and treatment of liver disease, screening and efficacy of drugs, cure condition, the degree of liver injury and forensic judgment, has very important application value of anti immune tolerance in liver transplantation rejection. With chemical purification method and it is very difficult to get a large number of F proteins, which hampered the other research. Therefore, cloning and expression of F protein in human liver by using gene engineering method, and induce the expression of F protein in human liver, to solve the problem. At the same time as source of genetic engineering protein for preliminary preparation monoclonal antibody and antigen specificity of this antibody was confirmed by immunoblotting, ELISA is the most suitable condition of next exploration, to lay the foundation for the study of immune tolerance and its immunological characteristics, Open a new door for the diagnosis and treatment of clinical liver disease.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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