EGFR基因重组噬菌体疫苗的构建及抗肿瘤效应的初步研究

发布时间：2018-06-25 04:05

本文选题：表皮生长因子受体 + T7噬菌体　；参考：《郑州大学》2005年硕士论文

【摘要】：目的:制备5个表达鸡源表皮生长因子受体(cEGFR)部分肽段的基因重组T7噬菌体疫苗,检测其EGFR抗原性,并初步观察其诱导产生的内源性抗EGFR抗体在C57BL/6J纯系小鼠Lewis肺癌模型上的抗肿瘤效果。方法:用RT-PCR方法从鸡睾丸组织总RNA中克隆出EGFR膜外区基因片段,将其克隆到载体pMD18-T Vector上,并转化E.coli DH5α感受态细胞,从阳性克隆中提取质粒,对该基因片段进行测序。用DNAStar软件对基因对应的蛋白质序列进行分析,选取亲水性高、抗原性强的区段,设计5对引物,将PCR扩增的片段亚克隆到T7噬菌体上。经筛选的5个肽段分别展示于其衣壳次要头蛋白(P10B)上,构建了5个cEGFR基因重组T7噬菌体疫苗。用该重组噬菌体侵染其宿主菌,液体扩增并纯化出重组噬菌体疫苗。经SDS-PAGE电泳及Western Blot抗原性检测,确认cEGFR-T7P10B融合蛋白的表达情况及其EGFR抗原性。用该重组噬菌体疫苗经脚掌、腋下及颌下皮内和颈背部皮下多点组合免疫4w龄的C57BL/6J纯系小鼠,每周1次,连续4w。免疫3w的小鼠血清与高表达EGFR的A431细胞孵育,抗鼠荧光二抗标记,流式细胞仪法检测被标记的细胞,确认特异性鼠抗EGFR抗体的产生。免疫4w后,腿部外侧皮下接种纯系Lewis肺癌细胞株,建立C57BL/6J纯系小鼠Lewis肺癌动物模型,10d后小心分离瘤体,各实验治疗组与空白噬菌体对照组瘤体均重两两比较采用t检验,观察各实验治疗组的抗肿瘤效果。
[Abstract]:Objective: to prepare five recombinant T7 phage vaccines expressing partial peptides of chicken epidermal growth factor receptor (cEGFR) and detect their EGFR antigenicity. The anti-tumor effect of endogenous anti-EGFR antibody induced by EGFR on C57BL / 6J mouse Lewis lung cancer model was also preliminarily observed. Methods: EGFR gene fragment was cloned from total RNA of chicken testis by RT-PCR, cloned into vector pMD18-T vector, transformed into E. coli DH5 伪 competent cells, and the plasmid was extracted from positive clones and sequenced. DNA Star software was used to analyze the corresponding protein sequence of the gene. Five pairs of primers were designed and subcloned into T7 phage by selecting the region with high hydrophilicity and strong antigenicity. Five peptides were screened on their capsid minor head protein (P10B) and five recombinant T7 phage vaccines with cEGFR gene were constructed. The recombinant bacteriophage was used to infect the host bacteria, and the recombinant phage vaccine was amplified and purified. The expression and antigenicity of cEGFR-T7P10B fusion protein were confirmed by SDS-PAGE and Western blot. The recombinant bacteriophage vaccine was used to immunize C57BL / 6J mice at the age of 4 weeks once a week for 4 weeks. The mouse serum was incubated with A431 cells with high EGFR expression. The labeled cells were detected by flow cytometry and the specific anti-EGFR antibody was detected by flow cytometry. After 4 weeks of immunization, Lewis lung cancer cell lines were inoculated subcutaneously on the lateral side of the leg, and the C57BL / 6J mouse Lewis lung cancer model was established. After 10 days of immunization, the tumor was carefully separated. The weight of the tumor in each experimental treatment group was compared with that in the blank phage control group by t test. To observe the anti-tumor effect of each experimental group.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392;R73-36

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