本文選題：TRX + 單克隆抗體��； 參考：《鄭州大學》2007年碩士論文

【摘要】： 丙型肝炎是感染丙型肝炎病毒(Hepatitis C virus，HCV)引起的傳染性疾病。全球估計約1.7億人感染HCV，中國感染人數(shù)約有3700萬。部分HCV感染者將發(fā)展成為慢性肝炎、肝硬化、肝細胞癌。因此，HCV的早期診斷顯得尤為重要。 HCV的實驗室診斷方法主要有：抗-HCV檢測、HCVAg檢測、重組免疫印跡分析(RIBA)、HCV RNA檢測等。其中，，抗HCV檢測試劑應用最為廣泛。目前主流的第三代抗HCV檢測試劑均采用間接ELISA，而間接ELISA普遍存在假陽性和漏檢的問題。與常規(guī)抗-HCV檢測方法相比，雙抗原夾心ELISA法可大大提高檢測的特異性和敏感性。不但能檢測到IgG抗體，而且還能夠檢測到早期出現(xiàn)的IgM抗體。然而雙抗原夾心ELISA試劑迄今尚未研制成功。其原因在于HCV抗原經(jīng)酶標記后，形成空間位阻，阻礙了抗體與抗原的結(jié)合。因此，直接標記抗原用于建立雙抗原夾心ELISA不容易成功。 硫氧還蛋白(thioredoxins，TRX)是分子量為12kDa的小分子蛋白質(zhì)，可在大腸桿菌中高度表達并具有高度親水性，能為丙肝抗原可溶性表達提供所必需的信號。據(jù)此制備出HCVAg-trx融合蛋白，若再以TRX作為橋蛋白，制備橋蛋白的單克隆抗體，用辣根過氧化物酶(HRP)標記。則由于橋蛋白及單抗的存在，標記的酶將不再阻礙抗原抗體的結(jié)合，能夠較好地解決位阻的問題。 目的 本課題針對現(xiàn)有丙型肝炎診斷試劑的缺陷，通過表達、純化TRX(橋蛋白)；制備TRX(橋蛋白)單克隆抗體，進行生物學鑒定。用辣根過氧化物酶(HRP)標記，為研制丙肝雙抗原夾心ELISA試劑奠定基礎(chǔ)。 方法 1．TRX的表達、純化將含有TRX基因的大腸桿菌經(jīng)IPTG誘導表達4小時，收集菌體，冰浴下超聲破菌，用親和層析法純化目的蛋白，12％SDS-PAGE鑒定。 2．雜交瘤細胞株的研制以純化TRX為抗原免疫8周齡的雌性BALB／c小鼠，三次免疫后，取免疫鼠脾細胞與SP2／0骨髓瘤細胞以6:1的比例融合。在HAT選擇培養(yǎng)基中選擇培養(yǎng)，用間接ELISA法篩選陽性細胞，有限稀釋法克隆和亞克隆，篩選出單克隆細胞株。 3．單克隆抗體的制備腹腔接種雜交瘤細胞，7-10天后采集腹水，并用正辛酸法以及離子層析純化，10％SDS-PAGE鑒定。 4．雜交瘤細胞及單克隆抗體的鑒定用秋水仙素阻抑法使細胞阻滯于分裂中期，顯微鏡下觀察，染色體計數(shù)，對雜交瘤細胞株進行染色體分析；采用間接ELISA測定單抗腹水效價、單抗相對親和力；采用間接ELISA和雙向免疫擴散法鑒定免疫球蛋白類型及亞類；以改良過碘酸鈉法制備酶標抗體并作偶聯(lián)物免疫活性的測定；以阻斷抑制法作TRX單抗識別抗原表位的測定。 5．TRX單抗與丙肝融合抗原(HCVAg-trx)反應測定以間接ELISA法和Western blotting鑒定單克隆抗體與丙肝融合抗原(HCVAg-trx)的特異性反應。 結(jié)果 1．TRX的表達、純化結(jié)果12％SDS-PAGE分析表明目的蛋白得到了高表達，分離效果較好，純度達到95％。 2．雜交瘤細胞株的研制獲得2株能穩(wěn)定分泌抗TRX單克隆抗體的雜交瘤細胞株，分別命名為2E8、5D6。 3．單克隆抗體的制備經(jīng)紫外分光光度計測定計算，2E8、5D6單抗的濃度分別為1.55mg／mL、0.839 mg／mL。10％SDS-PAGE結(jié)果顯示：純化單抗樣品在分子量約為150KD的位置出現(xiàn)有明顯的電泳條帶，為目的條帶，純度達到80％。 4．雜交瘤細胞及單克隆抗體的鑒定 (1)2E8、5D6的染色體平均條數(shù)分別為98、102，符合雜交瘤細胞染色體數(shù)約等于小鼠脾細胞和SP2／0骨髓瘤細胞染色體條數(shù)之和的理論值，由此證明所制備的細胞為雜交瘤細胞。 (2)兩株單抗腹水的效價均為1:10~7；相對親和力分析表明：5D6的相對親和力高于2E8。 (3)兩株單抗均為IgG類，IgG1亞類。 (4)2E8酶標抗體的免疫活性達到1:100×2~5，5D6酶標抗體達1:100×2~6。 (5)2E8和5D6兩株單抗相互阻斷，證明兩者識別的抗原表位顯著相關(guān)。 5．TRX單抗與重組丙肝抗原(HCVAg-trx)反應兩株單抗均能與TRX及HCVAg-trx發(fā)生特異性結(jié)合，而不與無關(guān)蛋白IFN-γ產(chǎn)生交叉反應。 結(jié)論 1．采用IPTG誘導表達，親和層析法純化，獲得高純度的重組TRX。 2．成功運用雜交瘤技術(shù)獲得兩株能穩(wěn)定分泌抗TRX的雜交瘤細胞株。 3．成功對雜交瘤細胞株和純化后的抗體進行了鑒定。 4．兩株單抗均能夠與丙肝融合抗原發(fā)生特異性結(jié)合反應。
[Abstract]:Hepatitis C is an infectious disease caused by hepatitis C virus ( HCV ) . Globally , about 170 million people have been infected with HCV , with a population of about 37 million . Some of the patients with HCV infection will develop into chronic hepatitis , cirrhosis and hepatocellular carcinoma . Therefore , early diagnosis of HCV is particularly important .









Anti - HCV detection , HCVAg detection , recombinant immune blotting analysis ( RIBA ) , HCV RNA detection , etc . The anti - HCV detection reagent is the most widely used .









Thioredoxins ( TRx ) is a small molecule protein with a molecular weight of 12 kDa , which can be highly expressed in E . coli and has high hydrophilicity . It can provide the necessary signal for the soluble expression of hepatitis C antigen .









Purpose









aiming at the defects of the prior hepatitis C diagnostic reagent , by expressing and purifying the trx ( bridge protein ) ;
Preparation of monoclonal antibody of trx ( bridge protein ) was carried out and biological identification was carried out . Using horseradish peroxidase ( HRP ) label , this paper lays a foundation for the development of HCV double antigen sandwich ELISA reagent .









method









1 . The expression of trx was purified and the E . coli containing trx gene was induced to express 4 hours by IPTG . The bacteria were collected , the ultrasonic bacteria were broken down under ice bath , the target protein was purified by affinity chromatography , and the 12 % SDS - PAGE was identified .









2 . The hybridoma cell line was prepared by immunizing eight - week old female BALB / c mice with purified trx . After three immunization , the spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells in a ratio of 6 : 1 . The positive cells were selected by indirect ELISA , the clones and subclones were screened by indirect ELISA , and the monoclonal cell lines were screened .









3 . The hybridoma cell was inoculated intraperitoneally with monoclonal antibody . The ascites was collected 7 - 10 days later and purified by means of n - octanol and ion chromatography . 10 % SDS - PAGE was used to identify the ascites .









4 . The hybridoma cell and the monoclonal antibody were used to identify the cell block in metaphase and metaphase , and the chromosome count was observed under the microscope and the chromosome analysis was carried out on the hybridoma cell line .
Indirect ELISA was used to determine the titer of monoclonal antibody and the relative affinity of monoclonal antibody .
Indirect ELISA and two - way immunodiffusion method were used to identify immunoglobulin type and subclass ;
preparing enzyme - labeled antibody by using modified sodium persulfate method and measuring the immunological activity of the conjugate ;
In this paper , the blocking suppression method was used to determine the epitope of the antigen epitope .









5 . The specific reaction of monoclonal antibody against hepatitis C fusion antigen ( HCVAg - trx ) was determined by indirect ELISA and Western blotting .









Results









1 . The results of SDS - PAGE analysis showed that the target protein was highly expressed , and the purity reached 95 % .









2 . The hybridoma cell lines secreting anti - trx monoclonal antibodies were obtained by the hybridoma cell line . The hybridoma cell lines were named 2e8 and 5D6 , respectively .









3 . The preparation of monoclonal antibody was determined by ultraviolet spectrophotometer . The concentration of the monoclonal antibody was 1.55 mg / mL and 0.839 mg / mL . 10 % SDS - PAGE showed that the purified monoclonal antibody had obvious electrophoretic bands at the position of about 150KD , and the purity reached 80 % .









4 . Identification of hybridoma cells and monoclonal antibodies









( 1 ) The average number of chromosome numbers of 2e8 and 5D6 was 98 , 102 , and the chromosome number of hybridoma cells was approximately equal to the theoretical value of chromosome number of mouse spleen cells and SP2 / 0 myeloma cells , thus demonstrating that the prepared cells were hybridoma cells .









( 2 ) the titer of the two monoclonal antibodies is 1 : 10 - 7 ;
The relative affinity analysis showed that the relative affinity of 5D6 was higher than that of 2E7 .









( 3 ) Both monoclonal antibodies were IgG , IgG1 subclass .









( 4 ) The activity of the enzyme labeled antibody of the enzyme was 1 : 100 脳 2 ~ 5 , the antibody of 5D6 enzyme was 1 : 100 脳 2 ~ 6 .









( 5 ) Two monoclonal antibodies were blocked from each other , which showed that the epitopes were significantly correlated with the epitope recognized by the two strains .









5 . Both monoclonal antibodies and recombinant hepatitis C antigen ( HCVAg - trx ) were able to bind specifically to trx and HCVAg - trx without cross - reaction with unrelated protein IFN - 緯 .









Conclusion









1 . IPTG - induced expression is adopted , affinity chromatography is adopted to purify , and high - purity recombinant trx is obtained .









2 . A hybridoma cell line secreting anti - trx stably was obtained by using hybridoma technique successfully .









3 . The hybridoma cell line and purified antibody were identified successfully .









4 . Both monoclonal antibodies can specifically bind to the hepatitis C fusion antigen .
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 胡翠華,李金明,王露楠,鄧巍;用不同的ELISA試劑盒檢測抗-HCV弱陽性血清標本的結(jié)果分析[J];實用肝臟病雜志;2004年03期

2 黃志強;丙型肝炎標志物的檢測研究進展及其臨床意義[J];國外醫(yī)學.臨床生物化學與檢驗學分冊;1994年03期

3 季陽 ,趙春紅;第三代重組免疫印跡試驗測定丙型肝炎病毒的可靠性[J];國外醫(yī)學.輸血及血液學分冊;1996年05期

4 王軍;在處于“窗口期”的抗體陰性的丙肝感染者中檢出丙肝核心抗原[J];國外醫(yī)學.輸血及血液學分冊;2001年02期

5 趙國勝,胡慶文;血清或血漿丙型肝炎病毒核心抗原作為血清學窗口期丙型肝炎病毒血癥標志物的檢測[J];國外醫(yī)學.輸血及血液學分冊;2002年06期

6 宋曉國,凌世淦,張賀秋,陳坤,孫柯兒,朱翠霞;重組丙型肝炎病毒表位抗原的研究[J];軍事醫(yī)學科學院院刊;2001年02期

7 楊利國,劉勝江;孕酮辣根過氧化物酶標記物的制備和鑒定[J];南京農(nóng)業(yè)大學學報;1986年03期

8 胡青,高大明,葉軍,武聚山,劉德恭;丙型肝炎的診斷治療現(xiàn)狀[J];中國全科醫(yī)學;2003年02期

9 葉群瑞,陳伯權(quán),吳美英;一種簡單、快速、量大的辛酸純化單克隆抗體法[J];生物化學與生物物理進展;1991年01期

10 陳浩,陳于紅,朱德煦,劉建寧;重組蛋白質(zhì)純化技術(shù)[J];中國生物工程雜志;2002年05期

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