本文選題：表皮生長因子受體 + T7噬菌體��； 參考：《鄭州大學(xué)》2005年碩士論文

【摘要】：目的:制備5個(gè)表達(dá)雞源表皮生長因子受體(cEGFR)部分肽段的基因重組T7噬菌體疫苗,檢測其EGFR抗原性,并初步觀察其誘導(dǎo)產(chǎn)生的內(nèi)源性抗EGFR抗體在C57BL/6J純系小鼠Lewis肺癌模型上的抗腫瘤效果。 方法:用RT-PCR方法從雞睪丸組織總RNA中克隆出EGFR膜外區(qū)基因片段,將其克隆到載體pMD18-T Vector上,并轉(zhuǎn)化E.coli DH5α感受態(tài)細(xì)胞,從陽性克隆中提取質(zhì)粒,對(duì)該基因片段進(jìn)行測序。用DNAStar軟件對(duì)基因?qū)?yīng)的蛋白質(zhì)序列進(jìn)行分析,選取親水性高、抗原性強(qiáng)的區(qū)段,設(shè)計(jì)5對(duì)引物,將PCR擴(kuò)增的片段亞克隆到T7噬菌體上。經(jīng)篩選的5個(gè)肽段分別展示于其衣殼次要頭蛋白(P10B)上,構(gòu)建了5個(gè)cEGFR基因重組T7噬菌體疫苗。用該重組噬菌體侵染其宿主菌,液體擴(kuò)增并純化出重組噬菌體疫苗。經(jīng)SDS-PAGE電泳及Western Blot抗原性檢測,確認(rèn)cEGFR-T7P10B融合蛋白的表達(dá)情況及其EGFR抗原性。用該重組噬菌體疫苗經(jīng)腳掌、腋下及頜下皮內(nèi)和頸背部皮下多點(diǎn)組合免疫4w齡的C57BL/6J純系小鼠,每周1次,連續(xù)4w。免疫3w的小鼠血清與高表達(dá)EGFR的A431細(xì)胞孵育,抗鼠熒光二抗標(biāo)記,流式細(xì)胞儀法檢測被標(biāo)記的細(xì)胞,確認(rèn)特異性鼠抗EGFR抗體的產(chǎn)生。免疫4w后,腿部外側(cè)皮下接種純系Lewis肺癌細(xì)胞株,建立C57BL/6J純系小鼠Lewis肺癌動(dòng)物模型,10d后小心分離瘤體,各實(shí)驗(yàn)治療組與空白噬菌體對(duì)照組瘤體均重兩兩比較采用t檢驗(yàn),觀察各實(shí)驗(yàn)治療組的抗腫瘤效果。
[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 偽 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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392;R73-36

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王桂云,朱筱娟,王麗;噬菌體展示外源多肽的免疫原性研究[J];東北師大學(xué)報(bào)(自然科學(xué)版);2001年03期

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本文編號(hào)：2064484