本文選題：人乳頭瘤病毒16型 + E6蛋白�。� 參考：《揚(yáng)州大學(xué)》2008年碩士論文

【摘要】： 人乳頭瘤病毒(HPV Human papillomavirus)是一組具有組織特異性的雙鏈DNA病毒。研究發(fā)現(xiàn),高危型HPV感染是宮頸癌發(fā)生發(fā)展的必要因素,特別是HPV16型在宮頸癌組織中檢出率可高達(dá)50%。HPV感染性疾病、宮頸癌已嚴(yán)重威脅了廣大婦女的生殖健康,現(xiàn)今條件下仍無特效的預(yù)防和治療方法。 乳頭瘤病毒基因組至少有10個(gè)開放閱讀框架(Open reading fame, ORF),主要位于同一條DNA鏈上,而另一條DNA鏈僅含少量的ORFs,被認(rèn)為是非編碼性的DNA鏈。各種HPV基因結(jié)構(gòu)大致相似,由早期(E)編碼區(qū)、晚期(L)編碼區(qū)以及介于E6和L1之間0.4 kb~1.01 kb的非編碼區(qū)3個(gè)部分組成。 本研究根據(jù)已發(fā)表的HPV16全基因組序列,設(shè)計(jì)并合成一對(duì)針對(duì)HPV16E6/7基因序列的特異性引物,以含HPV16E6/7蛋白基因的轉(zhuǎn)基因小鼠的癌組織DNA為模板,用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增HPV16E6/7基因序列,其大小為780 bp。 將PCR產(chǎn)物克隆入pGEM-T載體,構(gòu)建了重組質(zhì)粒pGEM-T-E6/7,經(jīng)EcoRI和XhoI限制性內(nèi)切酶雙酶切,回收目的片段,并克隆到PGEX-6P-1表達(dá)載體。通過PCR、酶切及測(cè)序鑒定證實(shí),HPV16E6/7基因以正確的閱讀框架插入到PGEX-6P-1載體中。將重組質(zhì)粒轉(zhuǎn)化至大腸桿菌BL21(DE3),經(jīng)IPTG誘導(dǎo)后,運(yùn)用SDS-PAGE電泳分析表達(dá)產(chǎn)物,并用Western Blot進(jìn)行鑒定,HPV16E6重組蛋白的相對(duì)分子質(zhì)量約為4.3×104。利用親和層析法從表達(dá)產(chǎn)物中純化出目的蛋白。 以純化的HPV16E6重組蛋白為免疫原免疫BALB/c小鼠,運(yùn)用淋巴細(xì)胞雜交瘤技術(shù)進(jìn)行細(xì)胞融合,并采用間接ELISA法進(jìn)行篩選,共獲得了4株能穩(wěn)定分泌針對(duì)HPV16E6蛋白的特異性單抗的雜交瘤細(xì)胞株,分別命名為4B11E1E8D4、5C2A4D6E8、4B12C5C8F7、2E3F9C4C6,抗體亞型檢測(cè)結(jié)果分別為IgG1、IgG2b、IgG1及IgG2a。利用間接ELISA、免疫印跡和間接免疫熒光試驗(yàn)鑒定,證實(shí)這4株單克隆抗體均能與重組HPV16E6蛋白發(fā)生特異性反應(yīng),其中5C2A4D6E8、4B12C5C8F7、2E3F9C4C6三株屬于相同的抗原表位,而4B11E1E8D4屬于不同抗原識(shí)別位點(diǎn)。本研究通過GST融合蛋白成功制備出針對(duì)人乳頭瘤病毒16E6蛋白的特異性單克隆抗體,為今后進(jìn)一步探索其生物學(xué)功能、HPV16E6的檢測(cè)以及免疫學(xué)治療奠定了基礎(chǔ)。
[Abstract]:Human papillomavirus (HPV) is a group of double strand DNA viruses with tissue specificity. The study found that high risk HPV infection is a necessary factor for the occurrence and development of cervical cancer, especially the detection rate of HPV16 type in cervical cancer tissue can be as high as 50.HPV infectious disease, cervical cancer has seriously threatened the reproductive health of women. There are still no effective methods for prevention and treatment under present conditions. There are at least 10 open reading frames (ORFs) in the genome of papillomavirus, mainly located on the same DNA strand, while the other strand contains only a small amount of ORFs, which is considered to be a non-coding DNA strand. The structures of HPVs are similar and consist of three parts: early (E) coding region, late (L) coding region and non-coding region of 0.4 kb~1.01 kb between E6 and L1. In this study, a pair of specific primers for HPV16 E6 / 7 gene sequence were designed and synthesized according to the published HPV16 full genome sequence. The HPV16 E6 / 7 gene sequence was amplified by polymerase chain reaction (PCR) from the cancer tissue DNA of transgenic mice containing HPV16 E6 / 7 protein gene, and the HPV16 E6 / 7 gene sequence was amplified by polymerase chain reaction (PCR). Its size is 780 BP. The PCR product was cloned into pGEM-T vector, and the recombinant plasmid pGEM-T-E6 / 7 was constructed. The recombinant plasmid pGEM-T-E6 / 7 was digested by EcoRI and XhoI restriction endonuclease, and the target fragment was recovered and cloned into PGEX-6P-1 expression vector. The HPV16E6 / 7 gene was inserted into PGEX-6P-1 vector by PCR, restriction endonuclease digestion and sequencing. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), induced by IPTG, the expressed product was analyzed by SDS-PAGE electrophoresis, and the relative molecular weight of HPV16E6 recombinant protein was identified as 4.3 脳 104 by Western blot. The target protein was purified from the expressed product by affinity chromatography. BALB / c mice were immunized with purified HPV16E6 recombinant protein. Lymphocyte hybridoma technique was used for cell fusion and indirect Elisa was used to screen BALB / c mice. Four hybridoma cell lines secreting specific monoclonal antibodies against HPV16E6 protein were obtained and named as 4B11E1E8D4C2A4D6E8A4B12C5C8F72E3F9C6C6. The antibody subtypes were IgG1 and IgG2a. Indirect Elisa, Western blot and indirect immunofluorescence assay showed that the four monoclonal antibodies could react specifically with recombinant HPV16E6 protein. Among them, 5C2A4D6E8E8A4B12C5C8F7O2E3F9C4C6 strains belong to the same epitope, while 4B11E1E8D4 belong to different antigen recognition sites. In this study, a specific monoclonal antibody against human papillomavirus 16E6 protein was successfully prepared by GST fusion protein, which laid a foundation for further exploring the biological function of HPV16E6 and immunological therapy.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 和燕玲;陳國華;王佩雅;薛萍;房永祥;景志忠;;豬IFN-α1重組蛋白單克隆抗體雜交瘤細(xì)胞株的建立及其生物學(xué)特性[J];中國獸醫(yī)科學(xué);2010年10期

相關(guān)碩士學(xué)位論文 前1條

1 陰秀麗;人乳頭瘤病毒16型E7蛋白單克隆抗體的研制[D];山東大學(xué);2011年

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本文編號(hào)：2103990