胃癌MG7-Ag模拟表位口服DNA疫苗的研制

目的　利用减毒鼠伤寒沙门氏菌研制胃癌MG7 Ag模拟表位的口服DNA疫苗 ,并观察其对小鼠的免疫效能及保护作用。方法　构建MG7 Ag模拟表位和通用性辅助性T细胞表位融合基因的真核表达载体。将真核表达载体转入减毒鼠伤寒沙门氏菌得到模拟表位的口服DNA疫苗。以 1× 10 8cfu疫苗菌口服免疫C5 7BL/6J小鼠 ,以携带空载体的沙门氏菌和PBS口服作为对照。以ELISA法检测小鼠血清中抗MG7 Ag抗体的滴度 ,以H TDR掺入法检测小鼠脾淋巴细胞对人工合成的MG7 Ag抗原肽刺激的增殖能力。同时 ,用表达MG7 Ag的小鼠艾氏腹水瘤细胞进行肿瘤攻击 ,观察疫苗对小鼠的保护作用。结果　口服疫苗可诱导小鼠产生MG7抗体 ,但各组小鼠脾淋巴细胞体外刺激增殖实验差异无显著性。肿瘤攻击 2周后 ,疫苗免疫组 7只小鼠中有 2只未见肿瘤形成 ,而对照组 4只小鼠则全部成瘤。结论　胃癌MG7 Ag模拟表位的口服DNA疫苗具有免疫原性 ,可以诱导小鼠产生抗肿瘤免疫 ,并具有一定保护作用     

MAGE-3多肽纳米疫苗对小鼠胃癌种植瘤抑瘤效应研究

背景与目的:纳米颗粒作为疫苗载体可保护抗原免受酶解,增强免疫原性,是一类极具开发潜力的新型疫苗载体。本实验制备负载CD4+CD8+T细胞表位MAGE-3多肽抗原的纳米疫苗,探讨其相关特性及抗肿瘤免疫。方法:利用自组装技术制备多肽/Chit-DC(壳聚糖-脱氧胆酸)载药纳米胶束,透射电镜观察纳米微观形态,荧光分光光度法计算负载率、载药量,并测定药物释放规律。流式细胞仪检测DC(树突状细胞)对药物的吞噬率,酶联免疫斑点实验(ELISPOT)和细胞毒性实验检测MAGE-3多肽纳米疫苗激活机体细胞免疫反应的状况。动物实验观察其体内抑瘤效应。结果:成功制备多肽/Chit-DC纳米胶束,药物包封率约为37%,载药量为17%。载药纳米颗粒中的多肽在pH7.4的PBS中释放缓慢,于48h达释放平台;在2mg/mL溶菌酶溶液中,药物有一定的突释现象,于24h达释放平台。ELISPOT和细胞毒性实验显示MAGE-3多肽纳米疫苗可以激活体内免疫反应而产生针对MAGE-3的CTL,特异性杀伤表达MAGE-3的肿瘤细胞。体内抑瘤实验显示,多肽纳米疫苗组相对肿瘤抑制率为37.81%。结论:MAGE-3多肽/Chit-DC纳米疫苗能有效激活体内的抗肿瘤免疫效应,抑制MFC小鼠前胃癌细胞的生长。     

血清骨桥蛋白、胃癌相关抗原和组织多肽特异性抗原诊断胃癌的临床价值

目的 探讨血清骨桥蛋白(OPN)、胃癌相关抗原(MG7-Ag)和组织多肽特异性抗原(TPS)对诊断胃癌的临床价值.方法 选择胃癌病例86例、癌前病变病例45例、胃良性病变病例48例与健康对照50例,采用ELISA法检测血清OPN、MG7-Ag与TPS.结果 胃癌组血清OPN、MG7-Ag和TPS水平及阳性率均高于其他3组,差异均有统计学意义(P<0.01).胃癌TNMⅢ/Ⅳ期病例、低分化胃癌病例及淋巴结转移的病例血清中OPN、MG7-Ag与TPS的水平分别高于Ⅰ/Ⅱ期、高/中分化及无淋巴结转移的病例(P均<0.05).OPN、MG7-Ag、TPS之间均呈显著的正相关关系(P均<0.05).结论 OPN、MG7-Ag与TPS在胃癌患者体内呈高表达,检测OPN、MG7-Ag、TPS对胃癌的早期诊断具有重要的意义.     

热休克蛋白72-甲胎蛋白抗原表位肽复合物抗小鼠肝癌Hepal-6细胞免疫的实验研究

目的:以热休克蛋白72(HSP72)-甲胎蛋白(AFP)抗原表位肽复合物免疫小鼠,研究该复合物针对AFP肿瘤是否具有特异性抗肿瘤免疫.方法:以HSP72-AFP多肽复合物皮下注射免疫昆明小鼠,并分别以AFP多肽和HSP72单独免疫小鼠为对照组.ELISA法检测免疫后小鼠血清IFN-γ水平、MTT法检测各免疫组小鼠淋巴细胞对肝癌Hepal-6细胞的杀伤作用、以小鼠体内瘤负荷实验评价蛋白复合物的免疫效应.结果:HSP72-AFP多肽复合物组小鼠血清IFN-γ水平、淋巴细胞对Hepal-6细胞的杀伤作用均显著高于AFP多肽和HSP72组(P＜0.01).HSP72-AFP多肽复合物组瘤体积也明显小于AFP多肽和HSP72免疫组(P＜0.01).结论:HSP72-AFP多肽复合物疫苗可诱导荷瘤小鼠产生针对AFP肿瘤的特异性细胞免疫,其对瘤细胞的杀伤效应明显优于两者单一的多肽纯化疫苗.表明HSP72-AFP多肽复合物可诱导小鼠产生有效的抗肿瘤免疫.     

血清MG7-Ag联合G-17检测在胃癌诊断中的临床价值

目的 探讨血清胃癌相关抗原(MG7-Ag)联合胃泌素-17(G-17)检测用于诊断胃癌的临床价值.方法 选择胃癌患者60例(胃癌组),胃部良性病变患者50例(良性病变组),癌前病变患者58例(癌前病变组),同时选择同期进行体检的健康志愿者50例作为对照组.检测所有患者以及健康对照组志愿者血清中MG7-Ag以及G-17的表达含量,分析2种检测指标用于诊断胃癌的临床效能.结果良性病变组、癌前病变组以及胃癌组患者血清中MG7-Ag以及G-17的表达含量显著高于健康对照组(P<0.05).胃癌进展期患者血清中MG7-Ag和G-17的含量水平显著高于早期胃癌患者(P<0.05);淋巴结转移患者血清中MG7-Ag和G-17的含量水平显著高于淋巴结非转移胃癌患者(P<0.05).与MG7-Ag、G-17单独用于诊断胃癌相比,MG7-Ag联合G-17用于诊断胃癌的敏感性、特异性更强,且阳性预测值和阴性预测值升高,其中,MG7-Ag联合G-17的敏感性、特异性,阳性预测值和阴性预测值均比MG7-Ag检测显著升高(P<0.05).结论MG7-Ag联合G-17用于诊断胃癌的特异度高,误诊率低,可以提高诊断准确性.     

噬菌体抗原模拟表位的免疫原性研究

目的 通过研究胃癌相关抗原的短肽模拟表位的免疫原性,探讨肿瘤抗原表位的噬菌体模拟肽是否具有免疫原性,为进一步的胃癌疫苗研制奠定实验基础。方法 以胃癌单克隆抗体MG7所识别的胃癌相关抗原表位模拟肽为免疫原,免疫Balb/c小鼠,获得免疫血清;用表达MG7单抗所识别的相应抗原的胃癌组织切片,通过免疫组化方法筛选免疫血清;在此基础上,对阳性血清进一步用细胞荧光标记法和ELISA法进行了检测,结果 免疫血清经免疫组化筛选和细胞荧光标记法及ELISA法检测,发现其中一个噬菌体呈现的表位模拟肽的免疫血清可与人胃癌组织间呈现特异的免疫反应,由此筛选出具有免疫原性的胃癌相关抗原表位的噬菌体模拟肽。结论 用单克隆抗体从随机肽库中筛选得到的部分表位模拟肽具有引发识别厮的始抗原免疫反应的免疫原性,提示以肿瘤相关抗原的表位模拟肽为基础研制疫菌具有可行性,可望在机体内诱发有效的体液免疫反应。     

负载胃癌新抗原肽和CD73抑制剂纳米粒子的中性粒细胞疫苗的制备和抗肿瘤效果评价

目的:制备负载胃癌细胞新抗原肽和CD73抑制剂的中性粒细胞（NEs）疫苗并表征,初步评价其在荷瘤615小鼠中的抗肿瘤效果。方法:通过Percoll梯度密度离心法分离纯化小鼠骨髓来源的NEs。分别制备仅负载胃癌细胞来源新抗原肽疫苗（DP-Ag-MFC-NEs）,以及同时负载新抗原肽和CD73抑制剂纳米粒子的联合疫苗（DP-Ag-MFC-NEs+antiCD73+CpG）,观察疫苗对荷瘤小鼠的治疗效果。结果:一只10周龄615小鼠可分离纯化到平均约1.5×106个NEs,细胞活性在95%以上,纯度在90%以上。和对照组相比,DP-Ag-MFC-NEs可显著抑制小鼠皮下肿瘤生长,使部分小鼠痊愈（CR）,小鼠淋巴结中CD4、CD8阳性细胞比例显著增高（P<0.05）。CR小鼠再次接种MFC胃癌细胞,没有小鼠肿瘤复发。联合疫苗的治疗效果确切,小鼠存活率最高（75%,P=0.038）。结论:负载胃癌细胞来源的新抗原肽和CD73抑制剂的中性粒细胞疫苗对小鼠无明显毒性,可显著抑制肿瘤生长,甚至治愈小鼠。同时,该疫苗可在小鼠体内激活记忆型免疫应答。     

血清与组织中MG7抗原表达对胃癌前病变风险预测的临床意义

背景与目的:多年来,许多科研人员致力于探索诊断胃癌及癌前疾病的生物学标志物.本研究通过对不同类型胃黏膜活检组织胃癌相关抗原(MG7-Ag)的表达和血清MG7-Ag含量的检测,探讨两者的相关性及对胃癌前病变风险预报的临床应用价值.方法:125例胃黏膜活检组织及其血清标本(正常胃黏膜12例,浅表性胃炎21例,胃黏膜糜烂溃疡24例,萎缩性胃炎15例,异型增生22例,胃癌31例),采用SP免疫组织化学二步法染色检测胃黏膜标本中MG7-Ag的表达情况;采用酶联免疫吸附实验检测血清MG7-Ag的含量.结果:①MG7-Ag在12例正常胃黏膜中无表达,在31例胃癌中表达率为93.55%,由浅表性胃炎(14.29%)或胃糜烂溃疡(33.33%)进展至萎缩性胃炎(86.67%)或异型增生(81.82%)再进展至胃癌(93.55%),MG7-Ag表达率依次逐渐上升,差异有显著性(P<0.05).②血清MG7-Ag含量从浅表性胃炎(3.0±0.6)、胃黏膜糜烂溃疡(2.8±2.0)、萎缩性胃炎(3.8±1.2)/异型增生(3.9±1.7)到胃癌组(7.0±4.6)有升高趋势,并且各组间比较差异均有非常显著性(P<0.01).胃癌患者血清MG7-Ag含量明显高于其他胃病患者,差异有显著性(P<0.05).③从浅表性胃炎到胃黏膜糜烂溃疡进展至萎缩性胃炎/异型增生再进展至胃癌,随着其病变组织MG7-Ag原位表达的上升,其血清MG7-Ag浓度有上升趋势,并且各组间比较差异均有非常显著性(P<0.01).两者具有良好的相关性(rs=0.346,P=0.001).结论:①MG7-Ag在胃疾病的动态表达提示胃黏膜细胞恶性程度与MG7-Ag的表达呈正相关,血清MG7-Ag与组织MG7-Ag表达有良好的相关性.②临床有望将MG7-Ag作为胃癌前病变随访,进行癌变风险预测以及早期诊断胃癌的预警标志物.③以血清标本代替组织标本检测UMG7-Ag具有取材方便,患者依从性好等优点,便于临床推广应用.     

人胃癌相关抗原MG7在胃癌前病变组织中的表达及其与COX-2表达的相关性分析

目的:探讨人胃癌相关抗原MG7在胃癌前病变组织中的表达及其与COX-2表达的相关性.方法: 采用常规ABC免疫组化染色法联合检测334例胃癌前病变患者的胃黏膜中MG7抗原和COX-2抗原的表达情况.结果: 在胃癌前病变组织(包括肠上皮化生和不典型增生)中MG7-Ag和COX-2的表达阳性率分别为49.1%和56.6%,从慢性浅表性胃炎、慢性萎缩性胃炎、肠上皮化生、不典型增生到胃癌MG7-Ag和COX-2的表达均呈上升趋势,差异具有显著性(P<0.05).MG7-Ag和COX-2在胃癌前病变组织中表达具有相关性.结论: MG7-Ag和COX-2在胃癌前病变组织中有一定的相关性.     

基于TEM-8新型颗粒性疫苗对结肠癌小鼠模型免疫治疗的作用

目的:用基于TEM-8(tumor endothelialmarker-8)真核表达的颗粒性疫苗免疫荷结肠癌小鼠,探讨该疫苗的免疫治疗作用。方法:用阳离子肽[K]16-tat49-57与带有负电荷的真核表达质粒结合成颗粒性疫苗,用透射电镜和免疫印迹法鉴定,酶联免疫斑点试验(ELISPOT)检测疫苗所诱导的特异性CTL分泌IFN-γ,并对荷瘤Balb/c小鼠进行免疫治疗后,观察荷瘤体积和小鼠生存率,用免疫组化方法(抗CD31)观察肿瘤组织血管生长情况,并计算肿瘤微血管密度。结果:在电荷比为r=2的制备条件下,所构建的颗粒性疫苗进行电镜扫描,显示疫苗能形成颗粒,转染COS-7细胞后可有效介导TEM-8蛋白表达。ELISPOT检测显示在体内疫苗能有效激发抗原特异性CTL效应,可诱导CTL产生IFN-γ等细胞因子,肿瘤治疗实验证实,颗粒性疫苗能有效抑制肿瘤的生长速度,提高荷瘤小鼠的生存率,与对照组相比差异有统计学意义,P<0.05。结论:基于TEM-8的新型抗肿瘤血管生成疫苗能有效激发出特异性CTL应答,并在荷瘤小鼠模型中有效抑制肿瘤血管生成,提高荷瘤小鼠的生存率,为新型结肠癌治疗性疫苗的设计提供了新思路。     

Enhanced immune response to gastric cancer specific antigen Peptide by coencapsulation with CpG oligodeoxynucleotides in nanoemulsion

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to construct a nanovaccine coencapsulated with a gastric cancer specific antigen MG7 mimotope peptide and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. It was shown that the tumor masses formed in the mice immunized with coencapsulated nanovaccine (0.0825 g) markedly smaller (P < 0.01) than those formed in the mice immunized with nanovaccine encapsulated with antigen peptide alone (0.4465 g). A tumor inhibiting rate as high as 82.5% of the coencapsulated nanovaccine was obtained, while nanovaccine encapsulated with peptide only could not achieve the same effect (28.5%) (P < 0.01). Enzyme-linked immunospot assay (ELISPOT) showed that immunization using MG7 mimotope peptide coencapsulated with CpG ODN within the same nanoemulsion enhanced the frequency of splenocytes secreting IFN-gamma significantly (P < 0.01) when compared with immunization using MG7 peptide encapsulated in nanoemulsion alone (197spots/1 x 10(6) vs. 73 spots/1 x 10(6)). Cellular ELISA indicated that serum titer of antibody against MG7-Ag was significantly higher (P < 0.01) in mice immunized with coencapsulation form nanovaccine (0.7884) than that in the group immunized with nanovaccine encapsulated with MG7 peptide alone (0.3616). Using intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD4+ T-cells. Our experiments suggest that a vaccinal approach using nano-delivery system carrying in tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.     

A novel DNA vaccine containing four mimicry epitopes for gastric cancer

Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.     

地方株HPV16E7基因疫苗诱发的小鼠免疫反应

目的评价地方株HPV16E7基因疫苗诱导的免疫反应。方法从先期构建的地方株HPV16E7基因重组质粒pGEM-T-E7经限制性核酸内切酶酶切,获得E7基因亚克隆到真核表达载体pcDNA3.1(-)中,构建地方株pcDNA-E7基因疫苗,转染CHO细胞,通过IFA试验验证E7蛋白的表达。用pcDNA-E7肌注方法免疫6周龄BALB/c小鼠,同时设pcDNA空载体为阴性对照,于第1天、第15天和第43天共免疫3次,用IFA和MTT法分析基因免疫在小鼠体内诱导的免疫应答。结果pcDNA-E7免疫小鼠后能检测出特异的抗体,而原载体免疫后不能产生特异抗体;但2组刺激淋巴细胞增殖的能力差异不显著。结论地方株HPV16E7基因疫苗可诱导产生小鼠免疫反应。     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

小鼠红白血病细胞来源的树突状细胞对特异性CTL的体内外诱导作用

目的 探讨白细胞介素-12(IL-12)诱导小鼠红白血病细胞产生的树突状细胞,对白血病特异性CTL细胞的诱导作用,及体内免疫后对抗肿瘤免疫应答的诱导效果.方法:采用4/小时~(51)Cr释放法,检测CTL杀伤活性;观察小鼠红白血病细胞来源的树突状细胞体内免疫冶疗后对肿瘤的抑制作用,采用了HE染色和电镜等技术,分析肿瘤组织的病理学变化.结果:IL-12诱导FBL-3细胞产生的DC,在体外可诱导出FBL-3细胞特异性的CTL.采用IL-12诱导FBL-3细胞产生的DC免疫C57 BL/6小鼠.体内诱导出的CTL杀伤活性明显高于对照组.实验组小鼠能够有效的抵抗野生型FBL-3细胞的再攻击;肿瘤生长受到明显的抑制,组织学观察显示,肿瘤局部有较多炎性细胞浸润和细胞坏死;透射电镜可观察到典型的凋亡征象,结论:IL-12诱导小鼠FBL-3红白血病细胞产生的树突状细胞,在体内外可诱导出FBL-3细胞特异性的CTL,体内免疫后可增强抗肿瘤免疫应答,该结果为白血病的免疫治疗提供了新的途径.     

血管内皮生长因子基因重组T7噬菌体肿瘤疫苗的免疫活性

目的:采用噬菌体展示技术,构建人血管内皮生长因子重组T7噬菌体疫苗,并检验疫苗诱导小鼠产生特异性抗体的免疫活性。方法:将人VEGF165基因与T7Select10-3b噬菌体基因重组,构建T7Select10-3b-VEGF噬菌体疫苗。将疫苗免疫C57BL/6J小鼠。免疫4周后,用ELISA法检测免疫小鼠血清中特异性抗VEGF抗体滴度。结果:疫苗组血清产生较高水平抗VEGF抗体。结论:异种血管内皮生长因子基因重组T7噬菌体疫苗可打破机体对自身VEGF的免疫耐受,诱导产生较高水平的特异性抗VEGF抗体。     

异种黑色素细胞疫苗诱导小鼠抗恶性黑色素瘤免疫反应

目的：研究异种黑色素细胞疫苗对小鼠恶性黑色素瘤生长的抑制新作用。方法制轩异种黑色素细胞疫苗,皮皮预防免疫小鼠后再接种恶性黑色素瘤,并以该疫苗皮下注射治疗恶性黑色素瘤鼠,随后测量肿瘤大小,采用^51Cr释放法,体外分析小鼠脾细胞的细胞毒性T细胞（CTL）活性,以间接ELISA法检测小鼠血清抗肿瘤抗 。结果预防免疫使90％小鼠恶性黑色素瘤生长受到抑制,疫苗治疗使50％小鼠肿瘤生长受到抑制,免疫组鼠较对照组CTL杀伤活性在20：1、10：1、5：1不同效靶比时,分别增高34．0％、24．1％及13．9％（P＜0．05）,体内出现抗恶性黑色素瘤抗体。结论：异种黑色素细胞疫羁可诱导小鼠抗恶性黑色素瘤特异性免疫反,抑制肿瘤生长。     

Anti-tumor activity and immune responses induced by human cancer-associated mucin core peptide

Objective: To investigate the immune responses induced by apomucin which is a mixture of mucin core peptide, in mice for elucidating the role of mucin core peptide in the modulation of cancers. Methods: Apomucin was isolated from human pancreatic cancer cell line SW1990. The mice were immunized with this apomucin (10μg/time×6) plus DETOX. Results: When immunized, all mice developed delayed-type hypersensitivity (DTH) after challenged with apomucin or synthetic peptide MUC-2 or MUC-3, while the mice immunized with apomucin alone did not develop DTH. No antibodies were detected by ELISA after immunization. When the spleen cells of vaccinated mice were cocultured with this apomucin (10–50μg/ml) and rhIL-2(50U/ml)in vitro, the proliferated lymphocytes showed cytotoxicity against human cancer cells, including colon cancer, gastric cancer, pancreatic cancer and leukemia as measured by Cr-51 release assay. Antibodies against MUC-2 and MUC-3 could block the cytotoxicity. Conclusion: It was identified that a vaccine combined of apomucin and immune adjuvant DETOX can induce cellular immune response and anti-tumor cytotoxicity in mice. This study was supported by National Natural Science Foundation of China No. 39570792.     

重组EGFR噬菌体疫苗抗肿瘤效果的研究

目的构建重组EGFR噬菌体疫苗并且观察其抗肿瘤效果。方法将鸡源EGFR膜外部分的5个基因片段插入到T7噬菌体展示系统中,EGFR蛋白以融合蛋白的形式展示在噬菌体的外壳蛋白10B上,从而构建成噬菌体疫苗。Western blot方法检测噬菌体壳蛋白上是否有EGFR表达。利用ELISA方法检测免疫小鼠血清中抗EGFR抗体。分离免疫小鼠脾细胞,检测淋巴细胞对靶细胞A431的细胞毒作用。C57小鼠经过4次免疫,接种Lewis肺癌,记录肿瘤的生长情况,评价疫苗的抗肿瘤效果。结果本实验成功构建5种鸡源EGFR噬菌体疫苗。Western blot。显示噬菌体壳蛋白上有EGFR表达。免疫后的小鼠血清中检测到抗EGFR抗体。体外分离脾细胞证明免疫后的小鼠脾细胞对EGFR受体高表达的A431细胞具有杀伤作用。靶细胞和效应细胞的比例在1:10时,最大杀伤率可达(45．74±7．21)％(P<0．001)。肿瘤生长曲线显示免疫后的C57小鼠Lewis肺癌生长受到明显的抑制。结论利用噬菌体展示技术构建的噬菌体疫苗是一种有效的抗肿瘤方法。