本文選題：腫瘤標志物 + 脫嘌呤/脫嘧啶核酸內(nèi)切酶��； 參考：《第三軍醫(yī)大學(xué)》2009年碩士論文

【摘要】： 腫瘤的早期發(fā)現(xiàn)和診斷對腫瘤的預(yù)防和治療至關(guān)重要。腫瘤標志物作為腫瘤臨床診斷的常規(guī)手段之一,對腫瘤的早期發(fā)現(xiàn)和療效觀察具有重要意義。隨著ELISA、RT-PCR、FISH、SELDI-TOF、蛋白質(zhì)組學(xué)、組織芯片和基因芯片等生物技術(shù)的飛速發(fā)展,使得許多具有應(yīng)用前景的腫瘤標志物逐一被發(fā)現(xiàn),但目前仍無任何一種腫瘤標志物可對惡性腫瘤進行準確診斷和預(yù)后預(yù)測,腫瘤的實驗室診斷尚缺乏高敏感性和高特異性的檢測方法。研究表明,現(xiàn)有腫瘤標志物存在特異性和陽性率低的缺點,不能很好達到腫瘤“早期診斷”這一目的。對腫瘤標志物進行聯(lián)合檢測能提高惡性腫瘤診斷的靈敏度及特異度,但仍存在診斷總準確率不高的缺陷。因此,尋找一種新的高敏感、高特異性的腫瘤標志物或建立一種新的腫瘤標志物的聯(lián)合檢測方法成為大多數(shù)實驗室研究的方向之一。 人脫嘌呤脫嘧啶核酸內(nèi)切酶(Human apurinic/apyrimidinic endonuclease/redox effector factor,hAPE1)是DNA損傷堿基切除修復(fù)(base excision repair, BER)途徑的關(guān)鍵酶,是一種多功能蛋白質(zhì),具有修復(fù)AP位點和氧化還原雙重功能,與細胞凋亡,腫瘤細胞的增殖、分化和轉(zhuǎn)化,放化療敏感性以及神經(jīng)系統(tǒng)退行性變化有著密切的關(guān)系。國內(nèi)外研究表明,hAPE1蛋白在機體各器官組織中均有表達,而腫瘤組織中的hAPE1定位和表達與相應(yīng)的正常組織有顯著差異,其表達水平與腫瘤放化療抵抗有關(guān)。本課題前期研究發(fā)現(xiàn),腫瘤患者血清中hAPE1蛋白表達顯著高于正常人。因此,hAPE1的表達水平(和/或類型)可作為篩選某些腫瘤的輔助手段,hAPE1有望成為腫瘤早期診斷中一項敏感而特異的指標。我們推測,對hAPE1進行血清學(xué)檢測可能有助于惡性腫瘤的早期臨床診斷,并有效判斷腫瘤細胞放療和化療的敏感性。 研究目的 1.構(gòu)建hAPE1原核表達載體,誘導(dǎo)表達、純化hAPE1融合蛋白,并對其生物學(xué)功能進行初步分析; 2.建立穩(wěn)定分泌hAPE1單克隆抗體的雜交瘤細胞株,制備并純化hAPE1單克隆抗體,鑒定其生物學(xué)特性并進行初步應(yīng)用; 3.初步應(yīng)用hAPE1血清學(xué)檢測方法探討hAPE1對惡性腫瘤的診斷作用,并與多腫瘤標志物蛋白芯片檢測方法比較,探討hAPE1和多腫瘤標志物蛋白芯片聯(lián)合檢測在惡性腫瘤診斷中的臨床意義。 研究內(nèi)容和方法 1. hAPE1原核表達載體的構(gòu)建、融合蛋白的純化及功能鑒定 通過分子克隆技術(shù)構(gòu)建包含hAPE1全長基因序列的pET28a-hAPE1原核表達載體,經(jīng)測序驗證后,將pET28a-hAPE1重組質(zhì)粒轉(zhuǎn)化入E.coli BL21(DE3),IPTG誘導(dǎo)表達并鑒定,以His親和層析技術(shù)純化蛋白,用Western blot及EMSA實驗分析hAPE1融合蛋白的生物學(xué)功能。 2. hAPE1單克隆抗體的制備、生物學(xué)特性鑒定及初步應(yīng)用 以純化的hAPE1融合蛋白為抗原四肢皮下和腹腔注射免疫Balb/c小鼠,應(yīng)用細胞融合、間接ELISA法篩選和克隆化技術(shù)獲得穩(wěn)定分泌hAPE1單克隆抗體的雜交瘤細胞株。以小鼠體內(nèi)誘生法產(chǎn)生腹水,鑒定單抗亞類后采用Protein G柱對其純化。采用秋水仙素阻抑實驗鑒定單抗的染色體核型,Western blot和間接ELISA法鑒定單抗特異性、種屬交叉反應(yīng)、效價和親和力,hAPE1 15肽陣列鑒定單抗的抗原表位。將單抗初步應(yīng)用于免疫印跡、免疫細胞化學(xué)、免疫細胞熒光和免疫組織化學(xué)試驗等實驗中評價單抗性能。 3. hAPE1雙抗夾心ELISA法在腫瘤血清學(xué)檢測中的初步應(yīng)用 對健康體檢者、良性病變患者、腎癌、前列腺癌和睪丸癌患者的C-12多腫瘤標志物蛋白芯片檢測結(jié)果進行回顧性分析,評價蛋白芯片的診斷作用;同時用hAPE1雙抗夾心ELISA法對同批樣本的血清標本進行檢測,統(tǒng)計分析hAPE1的血清含量和診斷評價,并對兩種檢測結(jié)果進行比較,評價hAPE1在惡性腫瘤診斷中的意義。 研究結(jié)果 1. hAPE1原核表達載體的構(gòu)建、融合蛋白的純化及功能鑒定 所構(gòu)建的pET28-hAPE1重組質(zhì)粒經(jīng)雙酶切及DNA測序鑒定表明構(gòu)建正確。pET28-hAPE1重組質(zhì)粒在37℃,0.4mmol/L IPTG的誘導(dǎo)條件下可在E.coli BL21(DE3)中高效表達,蛋白含量可達菌體總蛋白量的50%,經(jīng)SDS-PAGE及Western blot鑒定均在預(yù)期位置出現(xiàn)陽性條帶;通過His親和層析純化得到純度90%以上的hAPE1融合蛋白,經(jīng)SDS-PAGE、Western blot及EMSA鑒定hAPE1融合蛋白具有抗原性、AP位點修復(fù)活性和氧化還原活性。 2. hAPE1單克隆抗體的制備、生物學(xué)特性鑒定及初步應(yīng)用 經(jīng)過4次細胞融合,成功獲得了2株能穩(wěn)定分泌hAPE1單克隆抗體的雜交瘤細胞株,命名為雜交瘤細胞2-G1和4-F6,并通過抗體純化技術(shù)對大量制備的腹水進行濃縮和純化,得到純度90%以上的2-G1 mAb和4-F6 mAb。利用ELISA、Western blot等方法檢測其效價超過1:107,Ig亞類分別是IgG2b和IgG3,親和常數(shù)分別為1.8×10-7和3.4×10-5,能特異性識別hAPE1天然蛋白和融合蛋白,與兔、小鼠、大鼠無種屬交叉反應(yīng)。2-G1 mAb的抗原表位為構(gòu)象型表位,可應(yīng)用于免疫細胞化學(xué)、免疫細胞熒光和免疫組織化學(xué)等實驗技術(shù)中。 3. hAPE1雙抗夾心ELISA法在腫瘤血清學(xué)檢測中的初步應(yīng)用 健康體檢者、良性病變患者、腎癌、前列腺癌和睪丸癌患者血清中hAPE1含量分別為8.97 (2.47,13.58) ng/ml、9.54 ( 2.36,14.22) ng/ml、14.98 (7.14,45.33) ng/ml、14.29 (10.83,33.68) ng/ml和20.74 (8.76,58.81) ng/ml,腫瘤患者與健康體檢者比較差異有統(tǒng)計學(xué)意義( P 0.05 )。hAPE1雙抗夾心ELISA法對腎癌、前列腺癌和睪丸癌的診斷陽性率分別為:57.14%、53.33%和66.67%,C-12多腫瘤標志物蛋白芯片對腎癌、前列腺癌和睪丸癌的診斷陽性率分別為:48.65%、80.00%和62.16%。結(jié)果表明,兩種方法對3種腫瘤的診斷陽性率無統(tǒng)計學(xué)差異( P 0.05 )。除前列腺癌外,hAPE1與蛋白芯片聯(lián)合檢測腎癌和睪丸癌可顯著提高診斷陽性率,具有統(tǒng)計學(xué)差異( P 0.05 )。 結(jié)論 1.成功構(gòu)建hAPE1原核表達載體pET28a-hAPE1,可在E.coli BL21(DE3)中高效表達,經(jīng)His親和層析純化得到hAPE1融合蛋白,純度達90%以上,具有抗原性、AP位點修復(fù)活性及氧化還原活性。 2.成功獲得2株穩(wěn)定分泌抗hAPE1單克隆抗體的雜交瘤細胞株2-G1和4-F6,并純化獲得2株高純度、高效價、高特異性和較高親和力的hAPE1單克隆抗體2-G1 mAb和4-F6 mAb。其中2-G1 mAb的抗原表位為構(gòu)象型表位,可用于多種免疫學(xué)實驗中,是良好的檢測試劑。 3.腎癌、前列腺癌和睪丸癌的血清hAPE1蛋白水平顯著高于健康體檢者, hAPE1雙抗夾心ELISA法在惡性腫瘤診斷中具有臨床意義,與C-12多腫瘤標志物蛋白芯片聯(lián)合檢測可提高對腎癌和睪丸癌的診斷陽性率。
[Abstract]:The early detection and diagnosis of tumor are of great importance to the prevention and treatment of tumor . As one of the conventional methods for tumor clinical diagnosis , tumor markers have been found to be of great significance to the early detection and prognosis of tumor .


Human apurinic / apyrimidinic endonuclease ( hAPE1 ) is a key enzyme in DNA damage repair ( BER ) pathway . It is a multi - functional protein which has a close relationship with cell apoptosis , proliferation , differentiation and transformation of tumor cells , sensitivity to radiotherapy and chemotherapy .


Purpose of study


1 . Construction of the prokaryotic expression vector of hAPE1 , induced expression , purification of hAPE1 fusion protein , and preliminary analysis of its biological function ;


2 . A hybridoma cell line stably secreting hAPE1 monoclonal antibody is established , the hAPE1 monoclonal antibody is prepared and purified , the biological characteristics thereof are identified and the preliminary application is carried out ;


3 . The diagnostic role of hAPE1 in the diagnosis of malignant tumor was investigated by using the hAPE1 serological test method , and the clinical significance of the combination of hAPE1 and multi - tumor marker protein chip in the diagnosis of malignant tumor was discussed .


Research content and methods


1 . Construction of Prokaryotic Expression Vector of hAPE1 , Purification and Functional Identification of Fusion Protein


The prokaryotic expression vector pET28a - hAPE1 containing the full - length gene sequence of hAPE1 was constructed by molecular cloning . After sequencing , the pET28a - hAPE1 recombinant plasmid was transformed into E . coli BL21 ( DE3 ) . The protein was purified by IPTG affinity chromatography . Western blot and EMSA were used to analyze the biological function of hAPE1 fusion protein .


2 . Preparation , Characterization and Preliminary Application of Monoclonal Antibodies to hAPE1


A hybridoma cell line secreting hAPE1 monoclonal antibody was obtained by using purified hAPE1 fusion protein as antigen in subcutaneous and intraperitoneal injection of Balb / c mice . The hybridoma cell line stably secreting hAPE1 monoclonal antibody was obtained by means of cell fusion , indirect ELISA and cloning . The monoclonal antibody was identified by Western blot and indirect ELISA . The monoclonal antibody was identified by Western blot and indirect ELISA .


Preliminary Application of 3 . hAPE1 Double Anti - Sandwich ELISA in the Detection of Tumor Serology


The detection results of C - 12 multi - tumor marker protein in patients with healthy physical examination , benign lesion , renal cancer , prostate cancer and testicular cancer were analyzed retrospectively , and the diagnostic function of protein chip was evaluated ; meanwhile , the serum samples of the same batch of samples were tested by using the hAPE1 double anti - sandwich ELISA method , and the serum levels and diagnostic evaluation of hAPE1 were statistically analyzed , and the significance of hAPE1 in the diagnosis of malignant tumor was evaluated .


Results of the study


1 . Construction of Prokaryotic Expression Vector of hAPE1 , Purification and Functional Identification of Fusion Protein


The recombinant plasmid pET28 - hAPE1 was identified by double digestion and DNA sequencing . The recombinant plasmid pET28 - hAPE1 was highly expressed in E . coli BL21 ( DE3 ) at 37 鈩，

本文編號：1812653