本文選題：真核延伸因子2 + 單克隆抗體��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：本研究應(yīng)用蛋白質(zhì)組學(xué)方法,采用二維色譜和質(zhì)譜聯(lián)合應(yīng)用技術(shù),對直腸癌與癌旁組織進行分析,得到差異表達蛋白35個。通過多種參數(shù)分析及查閱大量文獻最終選定真核延伸因子2(e EF2)作為本研究的目標蛋白。接下來以e EF2氨基酸序列N端300個氨基酸為目標多肽,通過優(yōu)化密碼子合成目標基因片段,并成功克隆至原核表達載體p ET30a,將重組表達質(zhì)粒轉(zhuǎn)化感受態(tài)細胞,進行融合蛋白的誘導(dǎo)表達,并獲得了高純度的e EF2融合蛋白。以純化的e EF2融合蛋白免疫小鼠,取免疫小鼠的脾細胞與骨髓瘤細胞融合形成雜交瘤細胞,通過有限稀釋法篩選出四株穩(wěn)定分泌抗人e EF2單克隆抗體的雜交瘤細胞。單克隆抗體效價均達到1:240000以上,亞型均為Ig G型,抗體純度達90%以上。對四株e EF2單克隆抗體進行配對篩選,選出用于ELISA雙抗體夾心的最佳配對抗體,成功建立了e EF2雙抗體夾心ELISA檢測方法。最后我們應(yīng)用自建的雙抗體夾心ELISA方法檢測腫瘤患者血清中e EF2表達情況。本研究分為四部分,主要研究方法及結(jié)果如下:第一部分:二維色譜與質(zhì)譜聯(lián)合應(yīng)用分析直腸癌與癌旁組織的差異蛋白方法:1、取20例Dukes B期直腸癌腺癌的癌組織及癌旁組織標本,制備多肽混合物。2、應(yīng)用二維色譜和質(zhì)譜技術(shù)聯(lián)合分析癌組織及癌旁組織蛋白譜。3、通過數(shù)據(jù)分析得到直腸癌與癌旁組織差異表達蛋白。結(jié)果:1、癌組織:共獲得31618個多肽序列,經(jīng)整理鑒定得到813個蛋白。2、癌旁組織:共獲得23547個多肽序列,經(jīng)整理鑒定得到537個蛋白。3、經(jīng)統(tǒng)計分析,共獲得癌組織與癌旁組織的差異表達蛋白35個,其中在癌組織中上調(diào)表達的蛋白18個,下調(diào)表達蛋白17個。4、通過多種參數(shù)分析及查閱大量文獻最終選定真核延伸因子2(e EF2)作為本研究的目標蛋白。第二部分:e EF2基因的克隆及表達方法:1、分析e EF2蛋白氨基酸序列的抗原表位情況,確定N端300個氨基酸作為目標蛋白,并對密碼子進行優(yōu)化,使其更適合原核系統(tǒng)表達。2、應(yīng)用基因合成技術(shù)對優(yōu)化后的目的基因進行合成,在上游添加Nde I酶切位點,下游添加Xho I酶切位點。3、將合成的e EF2目的基因片段克隆至原核表達載體p ET30a中,構(gòu)建原核表達質(zhì)粒p ET30a-e EF2。4、將重組表達質(zhì)粒轉(zhuǎn)化E.coli BL21感受態(tài)細胞,進行融合蛋白的誘導(dǎo)表達。結(jié)果:1、成功構(gòu)建了原核表達質(zhì)粒p ET30a-e EF2。2、成功表達并分離、純化了e EF2融合蛋白。第三部分:e EF2單克隆抗體制備方法:1、免疫小鼠:以弗氏佐劑與純化的e EF2融合蛋白混合作為免疫原。2、雜交瘤細胞制備:取免疫小鼠的脾細胞與骨髓瘤細胞融合。3、雜交瘤細胞篩選:用ELISA方法測定效價,篩選出陽性細胞株。4、用有限稀釋法對篩選出的陽性雜交瘤細胞株進行抗體亞型鑒定、建株、冷凍保存。5、小鼠腹水誘導(dǎo):將陽性雜交瘤細胞株接種于小鼠腹腔制備腹水,檢測腹水抗體效價。6、e EF2單克隆抗體純化,亞型分析及效價檢測。7、ELISA雙抗體夾心法配對單克隆抗體篩選。結(jié)果:1、篩選出四株穩(wěn)定分泌抗人e EF2單克隆抗體的雜交瘤細胞株。2、單克隆抗體效價均達到1:240000以上,亞型均為Ig G型,抗體純度達90%以上。3、篩選出最佳的配對單克隆抗體,成功建立用于檢測樣本中e EF2含量的雙抗體夾心ELISA方法。第四部分:e EF2在腫瘤患者血清中的表達方法:1、以自制并篩選配對的e EF2單克隆抗體,用ELISA雙抗體夾心法檢測多種腫瘤患者血清中e EF2含量。2、通過購買商品化的磷酸化e EF2(p-e EF2)試劑盒檢測腫瘤患者血清中p-e EF2含量3、同時檢測健康體檢人群血清中e EF2及p-e EF2含量,作為正常對照。4、將腫瘤患者分為不同組別,包括治療前、化療中等,結(jié)合臨床資料分析e EF2及p-e EF2的變化規(guī)律,判斷不同腫瘤及腫瘤不同階段e EF2的變化規(guī)律。5、為了排除因標本留取時間不同對結(jié)果產(chǎn)生的影響,將2014年凍存和2015年凍存的標本進行分組比較。結(jié)果:1、e EF2在直腸癌治療前、直腸癌化療中、結(jié)腸癌、肺癌、乳腺癌患者血清中的檢測結(jié)果與健康對照組比較具有顯著差異。2、e EF2在胃癌與對照組、直腸癌治療前與直腸癌化療中的比較中則無顯著差異。3、p-e EF2在直腸癌治療前、直腸癌化療中患者血清中的檢測結(jié)果與健康對照組比較具有顯著差異。4、p-e EF2直腸癌治療前與直腸癌化療中的比較中也具有顯著差異。5、2014年凍存的標本和2015年凍存的標本進行分組比較,結(jié)果顯示兩組間e EF2及p-e EF2均無顯著差異。結(jié)論:1、應(yīng)用質(zhì)譜技術(shù)及蛋白質(zhì)組學(xué)研究,得到了直腸癌與癌旁組織的差異蛋白譜,明確了真核延伸因子2(e EF2)在直腸癌組織中上調(diào)表達。2、以N端300個氨基酸為目的片段的e EF2融合蛋白作為免疫原,成功制備了效價和特異性都很好的抗人e EF2單克隆抗體,并建立了e EF2雙抗體夾心ELISA檢測方法。說明e EF2蛋白N端有多個抗原表位,可誘導(dǎo)產(chǎn)生不同的單克隆抗體,為后續(xù)大量制備e EF2單抗指明了方向。3、通過對大量血清標本e EF2及p-e EF2的檢測結(jié)果分析,我們認為可將e EF2作為腫瘤標志物用于體檢腫瘤篩查,p-e EF2可作為敏感指標用于腫瘤治療過程的監(jiān)測。
[Abstract]:In this study, the proteomics method was used to analyze the rectal cancer and para cancer tissue by two dimensional chromatography and mass spectrometry, and 35 differentially expressed proteins were obtained. The target protein of this study was selected as the target protein of the eukaryotic extension factor 2 (E EF2) through a variety of parameters analysis and consulting a large number of documents. Then the e EF2 amino acid sequence was followed. N terminal 300 amino acid as the target polypeptide, by optimizing the codon to synthesize the target gene fragment, and successfully cloned the prokaryotic expression vector p ET30a, transforming the recombinant expression plasmid into the receptive cells, inducing the inducible expression of the fusion protein, and obtaining the high purity e EF2 fusion protein. The purified e EF2 fusion protein is immunized to mice and immunized small mice. Hybridoma cells were formed by fusion of spleen cells and myeloma cells, and four hybridoma cells secreting monoclonal antibodies against human e EF2 were screened by finite dilution method. The titer of monoclonal antibodies reached more than 1:240000, the subtype was Ig G and the purity of the antibody was over 90%. Four monoclonal antibodies against e EF2 were selected and selected to select the monoclonal antibodies. For the best paired antibody for ELISA double antibody sandwich, the e EF2 double antibody sandwich ELISA detection method was successfully established. Finally, we used the self built double antibody sandwich ELISA method to detect the expression of E EF2 in the serum of cancer patients. The study is divided into four parts. The main research methods and results are as follows: the first part: the combination of two-dimensional chromatography and mass spectrometry The differential protein method of rectal cancer and para cancer tissue was analyzed. 1, the polypeptide mixture.2 was prepared from 20 cases of cancer tissue and para cancer tissue of Dukes B rectal cancer. The protein spectrum of cancer tissue and para cancer tissue was analyzed by two-dimensional chromatograph and mass spectrometry, and the differential expression protein of rectal cancer and para cancer tissue was obtained by data analysis. Results: 1, cancer tissue: a total of 31618 polypeptide sequences were obtained, and 813 protein.2 were obtained by collation and identification. 23547 polypeptide sequences were obtained. 537 protein.3 were obtained by sorting and identification. Through statistical analysis, 35 proteins were obtained from cancer tissues and para cancerous tissues. Among them, 18 proteins expressed in cancer tissues were down regulated. DDA 17.4, through a variety of parameters analysis and consulting a large number of literature, the eukaryotic extension factor 2 (E EF2) was selected as the target protein of this study. The second part: the cloning and expression of E EF2 gene: 1, analyze the epitope of the amino acid sequence of the e EF2 protein, determine the 300 amino acids at the N end as the target protein, and enter the codon It is optimized to make it more suitable for the expression of.2 in the prokaryotic system. Using gene synthesis technology to synthesize the optimized target gene, add the Nde I enzyme cut site in the upstream and the downstream of the Xho I enzyme cutting site.3, and clone the synthesized e EF2 target gene fragment to the prokaryotic expression vector p ET30a, and construct the prokaryotic expression plasmid P ET30a-e. The expression plasmid transformed E.coli BL21 receptive cells to the induction expression of fusion protein. Results: 1, the prokaryotic expression plasmid P ET30a-e EF2.2 was successfully constructed and successfully expressed and separated, and the e EF2 fusion protein was purified. The third part: the preparation method of E EF2 monoclonal antibody: 1, the immunized mice mixed with the purified fusion e EF2 fusion protein. As immunogen.2, hybridoma cells were prepared: immunized mice spleen cells and myeloma cells fusion.3, hybridoma cells screening: ELISA method to determine titer, screening positive cell line.4, using the finite dilution method to identify the positive hybridoma cell strain of the screened positive hybridoma cell strain, frozen storage.5, mouse ascites induction: Yang The hybridoma cell line was inoculated in the abdominal cavity of mice to prepare ascites, detected the antibody titer of ascites.6, e EF2 monoclonal antibody purification, subtype analysis and titer detection.7, ELISA double antibody sandwich method paired monoclonal antibody screening. Results: 1, four hybridoma cell line.2, which secreted the anti human e EF2 monkline antibody, was screened and the monoclonal antibody titer was selected. All of them were above 1:240000, the subtype was Ig G, the purity of the antibody was over 90%.3, and the best paired monoclonal antibody was selected. The double antibody sandwich ELISA method used to detect the e EF2 content in the samples was successfully established. The fourth part: the expression of E EF2 in the sera of the tumor patients: 1, to self-made and screen the paired e EF2 monoclonal antibody and ELI The content of E EF2 in serum of multiple tumor patients was detected by SA double antibody sandwich method, and the content of P-E EF2 content in serum of cancer patients was detected by buying commercialized e EF2 (P-E EF2) kit. The content of E EF2 in the serum of healthy people was detected at the same time, and the tumor patients were divided into different groups, including pre treatment, as a normal control group. In the middle of chemotherapy, the changes of E EF2 and P-E EF2 were analyzed with clinical data, and the changes of E EF2 in different stages of tumor and tumor were judged. In order to exclude the effect of different time on the result, the specimens frozen in 2014 and frozen in 2015 were compared. Results: 1, e EF2 before rectal cancer treatment, rectal cancer In chemotherapy, the serum levels of colon, lung and breast cancer patients were significantly different from those in the healthy control group.2, e EF2 in gastric cancer and control group, there was no significant difference in the comparison of cancer before and after rectal cancer chemotherapy, and P-E EF2 before the treatment of rectal cancer, the detection results of serum in patients with rectal cancer chemotherapy and health were healthy. There was a significant difference in the comparison of.4, P-E EF2 before and after the chemotherapy of rectal cancer, there was a significant difference between the specimens of.52014 years frozen and the frozen specimens in 2015. The results showed that there was no significant difference between the two groups of E EF2 and P-E EF2. Conclusion: 1, the mass spectrometry and proteomics should be used. The differential protein spectrum of rectal cancer and paracancerous tissue was made clear that the eukaryotic extension factor 2 (E EF2) was up-regulated and expressed.2 in the rectal cancer tissue, and the e EF2 fusion protein of the 300 amino acids at the N terminal was used as the immunogen. The anti human e EF2 monoclonal antibody was successfully prepared and the e EF2 double antibody sandwich ELISA detection was established. It shows that there are multiple epitopes at the N end of the e EF2 protein, which can induce the production of different monoclonal antibodies and indicate the direction.3 for the subsequent large number of E EF2 monoclonal antibodies. Through the analysis of the detection results of E EF2 and P-E EF2 in a large number of serum specimens, we think that e EF2 can be used as a tumor marker for screening the tumor. The index is used to monitor the process of cancer treatment.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.37

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