直肠癌上调表达蛋白eEF2单克隆抗体制备及临床应用

发布时间：2018-07-01 09:02

本文选题：真核延伸因子2 + 单克隆抗体　；参考：《吉林大学》2016年博士论文

【摘要】：本研究应用蛋白质组学方法,采用二维色谱和质谱联合应用技术,对直肠癌与癌旁组织进行分析,得到差异表达蛋白35个。通过多种参数分析及查阅大量文献最终选定真核延伸因子2(e EF2)作为本研究的目标蛋白。接下来以e EF2氨基酸序列N端300个氨基酸为目标多肽,通过优化密码子合成目标基因片段,并成功克隆至原核表达载体p ET30a,将重组表达质粒转化感受态细胞,进行融合蛋白的诱导表达,并获得了高纯度的e EF2融合蛋白。以纯化的e EF2融合蛋白免疫小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞融合形成杂交瘤细胞,通过有限稀释法筛选出四株稳定分泌抗人e EF2单克隆抗体的杂交瘤细胞。单克隆抗体效价均达到1:240000以上,亚型均为Ig G型,抗体纯度达90%以上。对四株e EF2单克隆抗体进行配对筛选,选出用于ELISA双抗体夹心的最佳配对抗体,成功建立了e EF2双抗体夹心ELISA检测方法。最后我们应用自建的双抗体夹心ELISA方法检测肿瘤患者血清中e EF2表达情况。本研究分为四部分,主要研究方法及结果如下:第一部分:二维色谱与质谱联合应用分析直肠癌与癌旁组织的差异蛋白方法:1、取20例Dukes B期直肠癌腺癌的癌组织及癌旁组织标本,制备多肽混合物。2、应用二维色谱和质谱技术联合分析癌组织及癌旁组织蛋白谱。3、通过数据分析得到直肠癌与癌旁组织差异表达蛋白。结果:1、癌组织:共获得31618个多肽序列,经整理鉴定得到813个蛋白。2、癌旁组织:共获得23547个多肽序列,经整理鉴定得到537个蛋白。3、经统计分析,共获得癌组织与癌旁组织的差异表达蛋白35个,其中在癌组织中上调表达的蛋白18个,下调表达蛋白17个。4、通过多种参数分析及查阅大量文献最终选定真核延伸因子2(e EF2)作为本研究的目标蛋白。第二部分:e EF2基因的克隆及表达方法:1、分析e EF2蛋白氨基酸序列的抗原表位情况,确定N端300个氨基酸作为目标蛋白,并对密码子进行优化,使其更适合原核系统表达。2、应用基因合成技术对优化后的目的基因进行合成,在上游添加Nde I酶切位点,下游添加Xho I酶切位点。3、将合成的e EF2目的基因片段克隆至原核表达载体p ET30a中,构建原核表达质粒p ET30a-e EF2。4、将重组表达质粒转化E.coli BL21感受态细胞,进行融合蛋白的诱导表达。结果:1、成功构建了原核表达质粒p ET30a-e EF2。2、成功表达并分离、纯化了e EF2融合蛋白。第三部分:e EF2单克隆抗体制备方法:1、免疫小鼠:以弗氏佐剂与纯化的e EF2融合蛋白混合作为免疫原。2、杂交瘤细胞制备:取免疫小鼠的脾细胞与骨髓瘤细胞融合。3、杂交瘤细胞筛选:用ELISA方法测定效价,筛选出阳性细胞株。4、用有限稀释法对筛选出的阳性杂交瘤细胞株进行抗体亚型鉴定、建株、冷冻保存。5、小鼠腹水诱导:将阳性杂交瘤细胞株接种于小鼠腹腔制备腹水,检测腹水抗体效价。6、e EF2单克隆抗体纯化,亚型分析及效价检测。7、ELISA双抗体夹心法配对单克隆抗体筛选。结果:1、筛选出四株稳定分泌抗人e EF2单克隆抗体的杂交瘤细胞株。2、单克隆抗体效价均达到1:240000以上,亚型均为Ig G型,抗体纯度达90%以上。3、筛选出最佳的配对单克隆抗体,成功建立用于检测样本中e EF2含量的双抗体夹心ELISA方法。第四部分:e EF2在肿瘤患者血清中的表达方法:1、以自制并筛选配对的e EF2单克隆抗体,用ELISA双抗体夹心法检测多种肿瘤患者血清中e EF2含量。2、通过购买商品化的磷酸化e EF2(p-e EF2)试剂盒检测肿瘤患者血清中p-e EF2含量3、同时检测健康体检人群血清中e EF2及p-e EF2含量,作为正常对照。4、将肿瘤患者分为不同组别,包括治疗前、化疗中等,结合临床资料分析e EF2及p-e EF2的变化规律,判断不同肿瘤及肿瘤不同阶段e EF2的变化规律。5、为了排除因标本留取时间不同对结果产生的影响,将2014年冻存和2015年冻存的标本进行分组比较。结果:1、e EF2在直肠癌治疗前、直肠癌化疗中、结肠癌、肺癌、乳腺癌患者血清中的检测结果与健康对照组比较具有显著差异。2、e EF2在胃癌与对照组、直肠癌治疗前与直肠癌化疗中的比较中则无显著差异。3、p-e EF2在直肠癌治疗前、直肠癌化疗中患者血清中的检测结果与健康对照组比较具有显著差异。4、p-e EF2直肠癌治疗前与直肠癌化疗中的比较中也具有显著差异。5、2014年冻存的标本和2015年冻存的标本进行分组比较,结果显示两组间e EF2及p-e EF2均无显著差异。结论:1、应用质谱技术及蛋白质组学研究,得到了直肠癌与癌旁组织的差异蛋白谱,明确了真核延伸因子2(e EF2)在直肠癌组织中上调表达。2、以N端300个氨基酸为目的片段的e EF2融合蛋白作为免疫原,成功制备了效价和特异性都很好的抗人e EF2单克隆抗体,并建立了e EF2双抗体夹心ELISA检测方法。说明e EF2蛋白N端有多个抗原表位,可诱导产生不同的单克隆抗体,为后续大量制备e EF2单抗指明了方向。3、通过对大量血清标本e EF2及p-e EF2的检测结果分析,我们认为可将e EF2作为肿瘤标志物用于体检肿瘤筛查,p-e EF2可作为敏感指标用于肿瘤治疗过程的监测。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.37

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