本文關(guān)鍵詞：噬菌體展示技術(shù)篩選表皮生長因子受體抗原模擬表位　出處：《第四軍醫(yī)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 表皮生長因子受體 模擬表位 噬菌體肽庫 西妥昔單抗 尼妥珠單抗

【摘要】：結(jié)直腸癌已成為我國繼肺癌、胃癌、肝癌后的第四大最常見癌癥。既往研究發(fā)現(xiàn),表皮生長因子受體(epidermal growth factor receptor,EGFR)在結(jié)直腸癌中常出現(xiàn)過表達(dá),并進(jìn)一步通過其信號轉(zhuǎn)導(dǎo)引起腫瘤細(xì)胞增殖、血管生成、腫瘤侵襲和轉(zhuǎn)移能力增強(qiáng),細(xì)胞凋亡抑制,因此,EGFR已成為抗腫瘤藥物的一個(gè)理想靶點(diǎn)。經(jīng)FDA批準(zhǔn)上市,針對EGFR抗原表位研制的單克隆抗體藥物有尼妥珠單抗和西妥昔單抗。這兩株單抗在治療EGFR高表達(dá)的結(jié)直腸癌及其他腫瘤中,能專一的針對腫瘤細(xì)胞,減少對正常組織的損害,顯著提高患者的生存狀態(tài)。但其存在一定的不足,首先,分子量相對較大,降低了單抗藥物的治療效果,導(dǎo)致臨床使用效果不理想。其次,價(jià)格昂貴,且在治療過程中需長期使用,給病人及家屬帶來了較大的經(jīng)濟(jì)負(fù)擔(dān)。如果能將單抗藥物與其靶點(diǎn)結(jié)合的位點(diǎn)模擬出來,制成多肽疫苗,就可以避免單克隆抗體藥物的不足,大大提高治療效果,減輕患者的經(jīng)濟(jì)負(fù)擔(dān)。模擬抗原表位的方法有兩種,通過將噬菌體展示技術(shù)與傳統(tǒng)的抗原表位分析方法相比,我認(rèn)為噬菌體展示技術(shù)具有獨(dú)特的優(yōu)勢。噬菌體展示技術(shù)是將外源蛋白基因序列插入至噬菌體外殼蛋白,隨著噬菌體的重新組裝將外源蛋白融合在噬菌體的表面,其在抗原表位篩選中的優(yōu)點(diǎn)是既可識別并結(jié)合腫瘤抗原,又能感染宿主菌進(jìn)行擴(kuò)增。噬菌體隨機(jī)肽庫包含大量不同序列結(jié)構(gòu)的多肽,其可以作為任何靶抗原表位的模擬肽,通過肽庫對靶抗原進(jìn)行篩選時(shí),不需要空間構(gòu)象,只需靶抗原就可獲得具有抗原性和免疫原性的蛋白多肽,且反映了抗原結(jié)合位點(diǎn)的空間構(gòu)像。目前該技術(shù)在腫瘤的基礎(chǔ)研究以及治療新技術(shù)的開發(fā)中得到廣泛應(yīng)用。本實(shí)驗(yàn)擬用尼妥珠單抗及西妥昔單抗分別作為抗EGFR的靶標(biāo)蛋白,應(yīng)用噬菌體隨機(jī)十二肽庫對其抗原表位進(jìn)行探討分析。用兩株單克隆抗體藥物對同一靶點(diǎn)抗原表位進(jìn)行篩選分析,目前尚未有文獻(xiàn)報(bào)道。擬經(jīng)過三輪噬菌體的“吸附-洗脫-擴(kuò)增”的親和篩選實(shí)驗(yàn),獲得與單抗特異性結(jié)合的單克隆噬菌體,進(jìn)而對噬菌體提取單鏈DNA并測序,獲得抗EGFR抗體對應(yīng)抗原表位的氨基酸序列,對兩株單抗獲得的氨基酸序列進(jìn)行分析比較。采用競爭ELISA檢測方法,將篩選出高頻的單克隆噬菌體與EGFR高表達(dá)的caco2細(xì)胞膜蛋白提取液競爭結(jié)合西妥昔單抗及尼妥珠單抗,進(jìn)一步檢測所獲得的短肽與西妥昔單抗及尼妥珠單抗的親和活性,并對兩株單抗獲得短肽的親和性進(jìn)行分析比較,以尋找最具親和活性的短肽。目的1.利用噬菌體十二肽庫進(jìn)行生物淘洗,篩選表皮生長因子受體抗原模擬表位。2.篩選出的模擬短肽的生物活性檢測。方法以尼妥珠單抗及西妥昔單抗作為抗EGFR抗原的靶分子,在噬菌體十二肽庫中淘選表皮生長因子受體的抗原模擬表位,經(jīng)過3輪生物淘洗后,挑選單克隆噬菌斑進(jìn)行擴(kuò)增,用ELISA方法檢測,選擇與西妥昔單抗或尼妥珠單抗親和性較高的陽性單克隆噬菌體,對其進(jìn)行擴(kuò)增及測序。采用競爭ELISA的方法,對篩選出的高頻噬菌體單克隆與EGFR高表達(dá)的caco2細(xì)胞膜蛋白提取液競爭結(jié)合西妥昔單抗及尼妥珠單抗。結(jié)果1.以抗EGFR的單克隆抗體藥物西妥昔單抗作為靶標(biāo)蛋白,利用噬菌體隨機(jī)十二肽庫對靶蛋白進(jìn)行三輪生物淘洗,對每輪淘洗出來的噬菌體均進(jìn)行噬菌體滴度測定,測定結(jié)果可以看出,西妥昔單抗淘篩出來的噬菌體總體回收率從第一輪的(6.67×10-6)%增加到了第三輪的(2.80×10-3)%,可見通過三輪“吸附-洗脫-擴(kuò)增”的生物淘洗,噬菌體回收率得到了富集,富集率達(dá)到了420倍。用同樣的方法對尼妥珠單抗進(jìn)行淘洗,對淘洗結(jié)果進(jìn)行滴度測定,測定結(jié)果可以看出,尼妥珠單抗淘選出來的噬菌體總體回收率從第一輪的(8.67×10-6)%增加到了第三輪的(1.80×10-3)%,可見尼妥珠單抗通過三輪“吸附-洗脫-擴(kuò)增”的生物淘洗,噬菌體的回收率也得到富集,富集度為208倍。2.在第三輪測噬菌體滴度的平板中,分別隨機(jī)挑選30個(gè)分隔良好的單克隆藍(lán)斑進(jìn)行擴(kuò)增,擴(kuò)增后行滴度測定,以擴(kuò)增的單克隆噬菌體作為檢測組,以噬菌體原庫擴(kuò)增液作陰性對照組,進(jìn)行ELISA檢測。在陽性結(jié)果的評價(jià)中,以單克隆噬菌體擴(kuò)增液(檢測組)的OD值為噬菌體原庫擴(kuò)增液(陰性對照)的OD值的2倍以上視為陽性。以此標(biāo)準(zhǔn)對ELISA結(jié)果進(jìn)行分析,確定西妥昔單抗的陽性結(jié)果有17個(gè),而尼妥珠單抗的陽性結(jié)果有21個(gè),陽性率分別為56.67%和70%。這表明西妥昔單抗及尼妥珠單抗均淘篩出來了特異性結(jié)合的噬菌體。隨機(jī)挑選陽性噬菌體克隆進(jìn)行依賴性分析,可見隨著陽性噬菌體投入量的增加,其OD值也增大,說明其存在劑量依賴性。3.對檢測出來的陽性噬菌體克隆提取單鏈DNA,并進(jìn)行DNA序列測定,找出插入噬菌體的36個(gè)堿基,并將其翻譯成氨基酸,此即為噬菌體遞呈的12肽序列。對17個(gè)陽性的西妥昔單抗噬菌體進(jìn)行測序,有三段短肽頻率出現(xiàn)較高,分別為HSFKWLDSPRLR出現(xiàn)6次,HTSSLWHLFRST出現(xiàn)4次,HLFNHNKNLPKR出現(xiàn)3次。對21個(gè)陽性的尼妥珠單抗噬菌體進(jìn)行測序,出現(xiàn)兩段頻率較高的短肽,分別為HSFKWLDSPRLR出現(xiàn)8次,HWKSSYVKWHNV出現(xiàn)5次。其中西妥昔單抗和尼妥珠單抗有一共有序列HSFKWLDSPRLR。4.在競爭ELISA實(shí)驗(yàn)中,EGFR高表達(dá)的caco2細(xì)胞膜蛋白提取液可以與噬菌體單克隆HSFKWLDSPRLR,HTSSLWHLFRST,HLFNHNKNLPKR競爭性結(jié)合西妥昔單抗,三條短肽均出現(xiàn)不同程度的抑制。EGFR高表達(dá)的caco2細(xì)胞膜蛋白提取液與噬菌體單克隆HSFKWLDSPRLR,HWKSSYVKWHNV競爭性結(jié)合尼妥珠單抗,兩條短肽均出現(xiàn)不同程度的抑制,且共有序列HSFKWLDSPRLR的抑制率最高。結(jié)論初步利用西妥昔單抗及尼妥珠單抗可從噬菌體隨機(jī)肽庫中篩選到表皮生長因子受體相關(guān)抗原表位。EGFR高表達(dá)的caco2細(xì)胞膜蛋白提取液與篩選出的抗原模擬短肽可以競爭性結(jié)合西妥昔單抗及尼妥珠單抗。短肽HSFKWLDSPRLR可作為表皮生長因子受體的抗原模擬表位,為進(jìn)一步研制腫瘤疫苗奠定了基礎(chǔ)。
[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
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R73-36

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