噬菌体展示技术筛选表皮生长因子受体抗原模拟表位

发布时间：2017-12-27 09:46

本文关键词：噬菌体展示技术筛选表皮生长因子受体抗原模拟表位　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：结直肠癌已成为我国继肺癌、胃癌、肝癌后的第四大最常见癌症。既往研究发现,表皮生长因子受体(epidermal growth factor receptor,EGFR)在结直肠癌中常出现过表达,并进一步通过其信号转导引起肿瘤细胞增殖、血管生成、肿瘤侵袭和转移能力增强,细胞凋亡抑制,因此,EGFR已成为抗肿瘤药物的一个理想靶点。经FDA批准上市,针对EGFR抗原表位研制的单克隆抗体药物有尼妥珠单抗和西妥昔单抗。这两株单抗在治疗EGFR高表达的结直肠癌及其他肿瘤中,能专一的针对肿瘤细胞,减少对正常组织的损害,显著提高患者的生存状态。但其存在一定的不足,首先,分子量相对较大,降低了单抗药物的治疗效果,导致临床使用效果不理想。其次,价格昂贵,且在治疗过程中需长期使用,给病人及家属带来了较大的经济负担。如果能将单抗药物与其靶点结合的位点模拟出来,制成多肽疫苗,就可以避免单克隆抗体药物的不足,大大提高治疗效果,减轻患者的经济负担。模拟抗原表位的方法有两种,通过将噬菌体展示技术与传统的抗原表位分析方法相比,我认为噬菌体展示技术具有独特的优势。噬菌体展示技术是将外源蛋白基因序列插入至噬菌体外壳蛋白,随着噬菌体的重新组装将外源蛋白融合在噬菌体的表面,其在抗原表位筛选中的优点是既可识别并结合肿瘤抗原,又能感染宿主菌进行扩增。噬菌体随机肽库包含大量不同序列结构的多肽,其可以作为任何靶抗原表位的模拟肽,通过肽库对靶抗原进行筛选时,不需要空间构象,只需靶抗原就可获得具有抗原性和免疫原性的蛋白多肽,且反映了抗原结合位点的空间构像。目前该技术在肿瘤的基础研究以及治疗新技术的开发中得到广泛应用。本实验拟用尼妥珠单抗及西妥昔单抗分别作为抗EGFR的靶标蛋白,应用噬菌体随机十二肽库对其抗原表位进行探讨分析。用两株单克隆抗体药物对同一靶点抗原表位进行筛选分析,目前尚未有文献报道。拟经过三轮噬菌体的“吸附-洗脱-扩增”的亲和筛选实验,获得与单抗特异性结合的单克隆噬菌体,进而对噬菌体提取单链DNA并测序,获得抗EGFR抗体对应抗原表位的氨基酸序列,对两株单抗获得的氨基酸序列进行分析比较。采用竞争ELISA检测方法,将筛选出高频的单克隆噬菌体与EGFR高表达的caco2细胞膜蛋白提取液竞争结合西妥昔单抗及尼妥珠单抗,进一步检测所获得的短肽与西妥昔单抗及尼妥珠单抗的亲和活性,并对两株单抗获得短肽的亲和性进行分析比较,以寻找最具亲和活性的短肽。目的1.利用噬菌体十二肽库进行生物淘洗,筛选表皮生长因子受体抗原模拟表位。2.筛选出的模拟短肽的生物活性检测。方法以尼妥珠单抗及西妥昔单抗作为抗EGFR抗原的靶分子,在噬菌体十二肽库中淘选表皮生长因子受体的抗原模拟表位,经过3轮生物淘洗后,挑选单克隆噬菌斑进行扩增,用ELISA方法检测,选择与西妥昔单抗或尼妥珠单抗亲和性较高的阳性单克隆噬菌体,对其进行扩增及测序。采用竞争ELISA的方法,对筛选出的高频噬菌体单克隆与EGFR高表达的caco2细胞膜蛋白提取液竞争结合西妥昔单抗及尼妥珠单抗。结果1.以抗EGFR的单克隆抗体药物西妥昔单抗作为靶标蛋白,利用噬菌体随机十二肽库对靶蛋白进行三轮生物淘洗,对每轮淘洗出来的噬菌体均进行噬菌体滴度测定,测定结果可以看出,西妥昔单抗淘筛出来的噬菌体总体回收率从第一轮的(6.67×10-6)%增加到了第三轮的(2.80×10-3)%,可见通过三轮“吸附-洗脱-扩增”的生物淘洗,噬菌体回收率得到了富集,富集率达到了420倍。用同样的方法对尼妥珠单抗进行淘洗,对淘洗结果进行滴度测定,测定结果可以看出,尼妥珠单抗淘选出来的噬菌体总体回收率从第一轮的(8.67×10-6)%增加到了第三轮的(1.80×10-3)%,可见尼妥珠单抗通过三轮“吸附-洗脱-扩增”的生物淘洗,噬菌体的回收率也得到富集,富集度为208倍。2.在第三轮测噬菌体滴度的平板中,分别随机挑选30个分隔良好的单克隆蓝斑进行扩增,扩增后行滴度测定,以扩增的单克隆噬菌体作为检测组,以噬菌体原库扩增液作阴性对照组,进行ELISA检测。在阳性结果的评价中,以单克隆噬菌体扩增液(检测组)的OD值为噬菌体原库扩增液(阴性对照)的OD值的2倍以上视为阳性。以此标准对ELISA结果进行分析,确定西妥昔单抗的阳性结果有17个,而尼妥珠单抗的阳性结果有21个,阳性率分别为56.67%和70%。这表明西妥昔单抗及尼妥珠单抗均淘筛出来了特异性结合的噬菌体。随机挑选阳性噬菌体克隆进行依赖性分析,可见随着阳性噬菌体投入量的增加,其OD值也增大,说明其存在剂量依赖性。3.对检测出来的阳性噬菌体克隆提取单链DNA,并进行DNA序列测定,找出插入噬菌体的36个碱基,并将其翻译成氨基酸,此即为噬菌体递呈的12肽序列。对17个阳性的西妥昔单抗噬菌体进行测序,有三段短肽频率出现较高,分别为HSFKWLDSPRLR出现6次,HTSSLWHLFRST出现4次,HLFNHNKNLPKR出现3次。对21个阳性的尼妥珠单抗噬菌体进行测序,出现两段频率较高的短肽,分别为HSFKWLDSPRLR出现8次,HWKSSYVKWHNV出现5次。其中西妥昔单抗和尼妥珠单抗有一共有序列HSFKWLDSPRLR。4.在竞争ELISA实验中,EGFR高表达的caco2细胞膜蛋白提取液可以与噬菌体单克隆HSFKWLDSPRLR,HTSSLWHLFRST,HLFNHNKNLPKR竞争性结合西妥昔单抗,三条短肽均出现不同程度的抑制。EGFR高表达的caco2细胞膜蛋白提取液与噬菌体单克隆HSFKWLDSPRLR,HWKSSYVKWHNV竞争性结合尼妥珠单抗,两条短肽均出现不同程度的抑制,且共有序列HSFKWLDSPRLR的抑制率最高。结论初步利用西妥昔单抗及尼妥珠单抗可从噬菌体随机肽库中筛选到表皮生长因子受体相关抗原表位。EGFR高表达的caco2细胞膜蛋白提取液与筛选出的抗原模拟短肽可以竞争性结合西妥昔单抗及尼妥珠单抗。短肽HSFKWLDSPRLR可作为表皮生长因子受体的抗原模拟表位,为进一步研制肿瘤疫苗奠定了基础。
[Abstract]:Colorectal cancer has become the fourth most common cancer in China after lung cancer, gastric cancer and liver cancer. Previous studies have shown that epidermal growth factor receptor (epidermal growth factor receptor, EGFR) in colorectal cancer often appear over expression, and further through the signal transduction induced by enhanced tumor cell proliferation, angiogenesis, tumor invasion and metastasis, apoptosis inhibition, therefore, EGFR has become an ideal target for antitumor drugs the. The monoclonal antibody against EGFR antigen epitopes, approved by FDA, is naduzumab and cetuximab. These two McAbs can specifically target cancer cells, reduce damage to normal tissues and improve the survival status of EGFR in colorectal cancer and other tumors. However, there are some shortcomings, first of all, the molecular weight is relatively large, which reduces the therapeutic effect of McAbs and leads to the poor clinical use. Second, the price is expensive, and it needs to be used in the process of treatment for a long time, which brings a great economic burden to the patients and their families. If we can simulate the site of the combination of monoclonal antibody and its target, make peptide vaccine, it can avoid the shortage of monoclonal antibody drugs, greatly improve the treatment effect and reduce the economic burden of patients. There are two ways to mimic antigen epitope. By comparing phage display technology with traditional antigen epitope analysis method, I think phage display technology has unique advantages. Phage display technology is the exogenous protein gene sequence inserted into phage coat protein with phage reassemble the exogenous protein fusion on the phage surface, the antigen screening is the advantage of not only can recognize and bind to tumor antigens, and amplified the host bacteria infection. Phage random peptide library contains a large number of different sequence structure of peptides, which can be used to simulate any target peptide epitope, peptide library of target antigens by screening, not only need the space conformation, the target antigen can obtain protein polypeptides with antigenicity and immunogenicity, and reflects the antigen binding sites of space conformation. At present, the technology has been widely used in the basic research of tumor and the development of new treatment technology. The aim of this study is to use nntuzumab and cetuximab as target proteins against EGFR respectively. The phage displayed random peptide library was used to analyze the epitopes of twelve epitopes. The antigenic epitopes of the same target were screened and analyzed with two monoclonal antibody drugs, and no literature has been reported. After three rounds of quasi phage biopanning affinity screening experiments, obtain monoclonal phage binding with specific monoclonal antibody, and the extraction of single stranded DNA phage and sequenced to obtain the corresponding amino acid sequence of anti EGFR antibody epitope, amino acid sequence of two mAbs were analyzed and compared. The competition method of ELISA, the selected Caco2 cell membrane protein expression and monoclonal phage EGFR high-frequency extract compete with cetuximab and nimotuzumab, further testing the activity of affinity peptide and cetuximab and nimotuzumab, and two strains of monoclonal antibody was affinity short peptides were analyzed and compared, in order to find out the short peptide affinity activity. Objective 1. using phage twelve peptide library for biological cleaning and screening the mimic epitopes of epidermal growth factor receptor antigen (EGFR). 2. the biological activity detection of the simulated short peptide was screened. The method estimates for trastuzumab and cetuximab as molecular targets for anti EGFR antigen, epidermal growth factor receptor in panning twelve phage display peptide library mimotopes, after 3 rounds of biopanning, phage selected clones were amplified and detected by ELISA, the positive phage clone selection and cetuximab nimotuzumab or higher affinity, was amplified and sequenced. The competitive ELISA method was used to screen high frequency phage monoclonal and EGFR cell membrane protein extracts with high expression of Caco2, and compete for cetuximab and nmtuzumab. Results 1. with anti EGFR monoclonal antibody cetuximab as target protein, phage random twelve peptide library of target protein for each round of rounds of biopanning, the phage panning out were phage titer determination, determination results show that cetuximab resistant phage panning out overall recovery from the first round (6.67 x 10-6)% increased to three (2.80 x 10-3)%, visible through three rounds of "adsorption elution amplification" biopanning, phage recovery was enriched, enrichment ratio reached 420 times. Of nimotuzumab washing using the same method of washing results titer, test results can be seen, nimotuzumab panned out phage overall recovery from the first round of the (8.67 x 10-6)% increased to three (1.80 x 10-3)%, visible nimotuzumab by three biopanning of biopanning, phage recovery is also enriched, enrichment of 208 times. 2. in the third round of phage titer test, 30 separate blue spots were amplified and amplified. Then the titer was detected. The amplified phage displayed as the detection group, and the phage library as the negative control group. The ELISA was detected. In the evaluation of positive results in the amplification of liquid with the monoclonal phage (test group) the OD value of the original library of phage amplified liquid (negative control) was more than 2 times as positive. According to the analysis of ELISA results, 17 positive results of cetuximab were identified, while 21 of NP positive results were 56.67% and 70% respectively. This shows that both cetuximab and nritozumab all scout a specific binding phage. Random selection of positive phage clones
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-36

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