E6-GST融合蛋白表达及HPV16 E6单克隆抗体制备

发布时间：2018-07-06 20:28

本文选题：人乳头瘤病毒16型 + E6蛋白　；参考：《扬州大学》2008年硕士论文

【摘要】： 人乳头瘤病毒(HPV Human papillomavirus)是一组具有组织特异性的双链DNA病毒。研究发现,高危型HPV感染是宫颈癌发生发展的必要因素,特别是HPV16型在宫颈癌组织中检出率可高达50%。HPV感染性疾病、宫颈癌已严重威胁了广大妇女的生殖健康,现今条件下仍无特效的预防和治疗方法。乳头瘤病毒基因组至少有10个开放阅读框架(Open reading fame, ORF),主要位于同一条DNA链上,而另一条DNA链仅含少量的ORFs,被认为是非编码性的DNA链。各种HPV基因结构大致相似,由早期(E)编码区、晚期(L)编码区以及介于E6和L1之间0.4 kb~1.01 kb的非编码区3个部分组成。本研究根据已发表的HPV16全基因组序列,设计并合成一对针对HPV16E6/7基因序列的特异性引物,以含HPV16E6/7蛋白基因的转基因小鼠的癌组织DNA为模板,用聚合酶链式反应(PCR)扩增HPV16E6/7基因序列,其大小为780 bp。将PCR产物克隆入pGEM-T载体,构建了重组质粒pGEM-T-E6/7,经EcoRI和XhoI限制性内切酶双酶切,回收目的片段,并克隆到PGEX-6P-1表达载体。通过PCR、酶切及测序鉴定证实,HPV16E6/7基因以正确的阅读框架插入到PGEX-6P-1载体中。将重组质粒转化至大肠杆菌BL21(DE3),经IPTG诱导后,运用SDS-PAGE电泳分析表达产物,并用Western Blot进行鉴定,HPV16E6重组蛋白的相对分子质量约为4.3×104。利用亲和层析法从表达产物中纯化出目的蛋白。以纯化的HPV16E6重组蛋白为免疫原免疫BALB/c小鼠,运用淋巴细胞杂交瘤技术进行细胞融合,并采用间接ELISA法进行筛选,共获得了4株能稳定分泌针对HPV16E6蛋白的特异性单抗的杂交瘤细胞株,分别命名为4B11E1E8D4、5C2A4D6E8、4B12C5C8F7、2E3F9C4C6,抗体亚型检测结果分别为IgG1、IgG2b、IgG1及IgG2a。利用间接ELISA、免疫印迹和间接免疫荧光试验鉴定,证实这4株单克隆抗体均能与重组HPV16E6蛋白发生特异性反应,其中5C2A4D6E8、4B12C5C8F7、2E3F9C4C6三株属于相同的抗原表位,而4B11E1E8D4属于不同抗原识别位点。本研究通过GST融合蛋白成功制备出针对人乳头瘤病毒16E6蛋白的特异性单克隆抗体,为今后进一步探索其生物学功能、HPV16E6的检测以及免疫学治疗奠定了基础。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】