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稳定表达分泌型Zika NS1蛋白细胞株的构建及细胞永生化的探索

发布时间:2018-12-24 06:42
【摘要】:研究背景Zika病毒(Zika virus,ZIKV)感染已成为热带和亚热带地区重大的公共卫生问题。目前,尚没有特异性抗ZIKV药,疫苗也仍处于研发阶段。早期诊断在防止ZIKV传染中具有重要的意义,在急性感染阶段,病毒编码NS1蛋白并释放入血。不同黄病毒的NS1蛋白高度保守,因此被用于鉴别诊断,NS1蛋白是用于血清学诊断ZIKV的首选蛋白。永生化细胞PER.C6由Crucell公司通过把人5型腺病毒E1区编码基因转染人的18周龄胎儿成视网膜细胞(HER)而获得。该细胞系具高效、稳定表达外源基因能力,在疫苗、重组蛋白、单克隆抗体和基因治疗等方面产品的生产得到广泛应用。然而,昂贵的转让费用也使得多数生物制药公司望而却步。目的本研究旨在建立ZIKV NS1蛋白哺乳细胞表达系统,获得高质量NS1抗原,为ZIKV血清学检测奠定基础。本研究还初步探索了永生化细胞系的建立方法,以期获得更具优势的哺乳动物细胞表达工具。方法将ZIKV NS1表达基因克隆至慢病毒表达载体中,鉴定正确的重组质粒与辅助质粒共转染HEK293T细胞,48h收集慢病毒(LV-CMV-EGFP-Zika-NSl),将LV-CMV-EGFP-Zika-NS1加入HEK 293T细胞中,挑选重组单克隆细胞。分别采用Western Blot和流式细胞分析检测连续传代的重组细胞株上清分泌NS1蛋白水平和细胞荧光率。亲和层析纯化重组蛋白,SDS-PAGE分析纯化效果。将纯化的NS1蛋白免疫BALB/c小鼠,间接ELISA测定其血清抗体水平。构建腺病毒E1基因表达载体pUC18-PGK-E1-polyA,转染L02细胞,48 h后对转染的细胞进行1:100倍稀释,连续培养20 d,挑选与原始细胞有形态学差异的细胞灶进行Western Blot鉴定。结果酶切和测序结果表明,成功构建了 pLV-CMV-EGFP-Zika-NSl重组表达载体。包装慢病毒感染HEK 293T细胞,采用有限稀释法获得8株重组表达ZIKVNS1的单克隆HEK 293T细胞株。Western Blot结果表明其中第7号克隆表达水平最高,将其命名为HEK-293T-Zika-NSl。Western Blot及流式细胞分析结果表明重组细胞株连续传代至第35代其NS1蛋白表达水平、荧光率和荧光强度均未发生显著变化。纯化的rNSl纯度超过90%。纯化的rNS1加强免疫BALB/c小鼠后,生成高水平的抗NS1抗体。Western Blot结果表明转染pUC18-PGK-E1-polyA质粒的L02细胞表达腺病毒E1蛋白,转染的L02细胞能观察到与原始细胞有形态学差异的细胞灶。通过Western Blot鉴定目前尚未筛选到表达Ad5 E1蛋白的细胞灶。结论本研究成功构建了重组表达ZIKVNS1蛋白的HEK293T细胞系,获得高纯度的重组NS1蛋白。该抗原具有较好免疫原性,刺激小鼠机体产生高水平抗体。该研究为ZIKV血清学诊断方法的奠定基础,此外建立的重组细胞系为ZIKV NS1蛋白的生物学特性研究提供了细胞模型。通过质粒及慢病毒转染方法制备永生化细胞均尚未获得成功,需要对现有方法进行改进或探索新的细胞永生化制备方法。
[Abstract]:Background Zika virus (Zika virus,ZIKV) infection has become a major public health problem in tropical and subtropical regions. At present, there is no specific anti-ZIKV drug, and the vaccine is still in the development stage. Early diagnosis plays an important role in preventing ZIKV infection. In acute infection stage, the virus encodes NS1 protein and releases it into blood. The NS1 proteins of different yellow viruses are highly conserved, so they are used in differential diagnosis, and NS1 protein is the first choice for serological diagnosis of ZIKV. PER.C6 of immortalized cells was obtained by transfection of human adenovirus E1 coding gene into human 18 week-old fetal retinal progenitor cells (HER) by Crucell Company. The cell line has the ability to express foreign gene stably and has been widely used in vaccine, recombinant protein, monoclonal antibody and gene therapy. However, the high transfer costs have deterred most biopharmaceutical companies. Objective to establish a lactation cell expression system of ZIKV NS1 protein and obtain high quality NS1 antigen, and to lay a foundation for ZIKV serological detection. This study also explored the establishment of immortalized cell lines in order to obtain more advantageous mammalian cell expression tools. Methods ZIKV NS1 expression gene was cloned into lentivirus expression vector, and the correct recombinant plasmid was cotransfected into HEK293T cells. Lentivirus (LV-CMV-EGFP-Zika-NSl) was collected after 48 hours. LV-CMV-EGFP-Zika-NS1 was added into HEK 293T cells and recombinant monoclonal cells were selected. Western Blot and flow cytometry were used to detect the level of NS1 protein and the fluorescence rate of the supernatant. The recombinant protein was purified by affinity chromatography and purified by SDS-PAGE. The purified NS1 protein was immunized with BALB/c mice and its serum antibody level was determined by indirect ELISA. Adenovirus E1 gene expression vector (pUC18-PGK-E1-polyA,) was constructed and transfected into L02 cells. After 48 hours, the transfected cells were diluted by 1: 100 times and cultured for 20 days. The foci with morphological difference from the original cells were identified by Western Blot. Results restriction endonuclease digestion and sequencing showed that the recombinant expression vector of pLV-CMV-EGFP-Zika-NSl was successfully constructed. HEK 293T cells were infected with lentivirus, and 8 monoclonal HEK 293T cell lines expressing ZIKVNS1 were obtained by limited dilution method. The results showed that the expression level of clone 7 was the highest. The results of HEK-293T-Zika-NSl.Western Blot and flow cytometry showed that the expression level of NS1 protein in the recombinant cell line was not significantly changed to the 35th generation. The fluorescence rate and fluorescence intensity of the recombinant cell line did not change significantly. The purity of purified rNSl was more than 90%. After BALB/c mice were immunized with purified rNS1, a high level of anti NS1 antibody. Western Blot was produced. The results showed that L02 cells transfected with pUC18-PGK-E1-polyA plasmid expressed adenovirus E1 protein. The transfected L02 cells were able to observe morphologically different foci from the original cells. The cell foci expressing Ad5 E1 protein have not been screened by Western Blot. Conclusion the recombinant HEK293T cell line expressing ZIKVNS1 protein was successfully constructed and a high purity recombinant NS1 protein was obtained. The antigen has good immunogenicity and stimulates the production of high-level antibodies in mice. This study laid a foundation for the serological diagnosis of ZIKV, and provided a cell model for the study of the biological characteristics of ZIKV NS1 protein. The immortalized cells prepared by plasmid and lentivirus transfection methods have not been successfully prepared, so it is necessary to improve the existing methods or to explore new methods of immortalization preparation.
【学位授予单位】:南方医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R373

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