穩(wěn)定表達(dá)分泌型Zika NS1蛋白細(xì)胞株的構(gòu)建及細(xì)胞永生化的探索
發(fā)布時(shí)間：2018-12-24 06:42
【摘要】：研究背景Zika病毒(Zika virus,ZIKV)感染已成為熱帶和亞熱帶地區(qū)重大的公共衛(wèi)生問(wèn)題。目前,尚沒(méi)有特異性抗ZIKV藥,疫苗也仍處于研發(fā)階段。早期診斷在防止ZIKV傳染中具有重要的意義,在急性感染階段,病毒編碼NS1蛋白并釋放入血。不同黃病毒的NS1蛋白高度保守,因此被用于鑒別診斷,NS1蛋白是用于血清學(xué)診斷ZIKV的首選蛋白。永生化細(xì)胞PER.C6由Crucell公司通過(guò)把人5型腺病毒E1區(qū)編碼基因轉(zhuǎn)染人的18周齡胎兒成視網(wǎng)膜細(xì)胞(HER)而獲得。該細(xì)胞系具高效、穩(wěn)定表達(dá)外源基因能力,在疫苗、重組蛋白、單克隆抗體和基因治療等方面產(chǎn)品的生產(chǎn)得到廣泛應(yīng)用。然而,昂貴的轉(zhuǎn)讓費(fèi)用也使得多數(shù)生物制藥公司望而卻步。目的本研究旨在建立ZIKV NS1蛋白哺乳細(xì)胞表達(dá)系統(tǒng),獲得高質(zhì)量NS1抗原,為ZIKV血清學(xué)檢測(cè)奠定基礎(chǔ)。本研究還初步探索了永生化細(xì)胞系的建立方法,以期獲得更具優(yōu)勢(shì)的哺乳動(dòng)物細(xì)胞表達(dá)工具。方法將ZIKV NS1表達(dá)基因克隆至慢病毒表達(dá)載體中,鑒定正確的重組質(zhì)粒與輔助質(zhì)粒共轉(zhuǎn)染HEK293T細(xì)胞,48h收集慢病毒(LV-CMV-EGFP-Zika-NSl),將LV-CMV-EGFP-Zika-NS1加入HEK 293T細(xì)胞中,挑選重組單克隆細(xì)胞。分別采用Western Blot和流式細(xì)胞分析檢測(cè)連續(xù)傳代的重組細(xì)胞株上清分泌NS1蛋白水平和細(xì)胞熒光率。親和層析純化重組蛋白,SDS-PAGE分析純化效果。將純化的NS1蛋白免疫BALB/c小鼠,間接ELISA測(cè)定其血清抗體水平。構(gòu)建腺病毒E1基因表達(dá)載體pUC18-PGK-E1-polyA,轉(zhuǎn)染L02細(xì)胞,48 h后對(duì)轉(zhuǎn)染的細(xì)胞進(jìn)行1:100倍稀釋,連續(xù)培養(yǎng)20 d,挑選與原始細(xì)胞有形態(tài)學(xué)差異的細(xì)胞灶進(jìn)行Western Blot鑒定。結(jié)果酶切和測(cè)序結(jié)果表明,成功構(gòu)建了 pLV-CMV-EGFP-Zika-NSl重組表達(dá)載體。包裝慢病毒感染HEK 293T細(xì)胞,采用有限稀釋法獲得8株重組表達(dá)ZIKVNS1的單克隆HEK 293T細(xì)胞株。Western Blot結(jié)果表明其中第7號(hào)克隆表達(dá)水平最高,將其命名為HEK-293T-Zika-NSl。Western Blot及流式細(xì)胞分析結(jié)果表明重組細(xì)胞株連續(xù)傳代至第35代其N(xiāo)S1蛋白表達(dá)水平、熒光率和熒光強(qiáng)度均未發(fā)生顯著變化。純化的rNSl純度超過(guò)90%。純化的rNS1加強(qiáng)免疫BALB/c小鼠后,生成高水平的抗NS1抗體。Western Blot結(jié)果表明轉(zhuǎn)染pUC18-PGK-E1-polyA質(zhì)粒的L02細(xì)胞表達(dá)腺病毒E1蛋白,轉(zhuǎn)染的L02細(xì)胞能觀察到與原始細(xì)胞有形態(tài)學(xué)差異的細(xì)胞灶。通過(guò)Western Blot鑒定目前尚未篩選到表達(dá)Ad5 E1蛋白的細(xì)胞灶。結(jié)論本研究成功構(gòu)建了重組表達(dá)ZIKVNS1蛋白的HEK293T細(xì)胞系,獲得高純度的重組NS1蛋白。該抗原具有較好免疫原性,刺激小鼠機(jī)體產(chǎn)生高水平抗體。該研究為ZIKV血清學(xué)診斷方法的奠定基礎(chǔ),此外建立的重組細(xì)胞系為ZIKV NS1蛋白的生物學(xué)特性研究提供了細(xì)胞模型。通過(guò)質(zhì)粒及慢病毒轉(zhuǎn)染方法制備永生化細(xì)胞均尚未獲得成功,需要對(duì)現(xiàn)有方法進(jìn)行改進(jìn)或探索新的細(xì)胞永生化制備方法。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R373
本文編號(hào)：2390276
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R373
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