{"title":"[干扰素-lambda1 在 CHO 细胞中的表达]。","authors":"Wu-Mei Yuan, Fen-Lian Ma, Qian Zhang, Wen-Zhi Zheng, Li-Shu Zheng","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells.</p><p><strong>Methods: </strong>Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1.</p><p><strong>Results: </strong>Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity.</p><p><strong>Conclusion: </strong>Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.</p>","PeriodicalId":70973,"journal":{"name":"中华实验和临床病毒学杂志","volume":"27 3","pages":"190-2"},"PeriodicalIF":0.0000,"publicationDate":"2013-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"[The expression of interferon-lambda1 in CHO cell].\",\"authors\":\"Wu-Mei Yuan, Fen-Lian Ma, Qian Zhang, Wen-Zhi Zheng, Li-Shu Zheng\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells.</p><p><strong>Methods: </strong>Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1.</p><p><strong>Results: </strong>Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity.</p><p><strong>Conclusion: </strong>Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.</p>\",\"PeriodicalId\":70973,\"journal\":{\"name\":\"中华实验和临床病毒学杂志\",\"volume\":\"27 3\",\"pages\":\"190-2\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2013-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中华实验和临床病毒学杂志\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中华实验和临床病毒学杂志","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
摘要
目的构建真核表达载体PCI-dhfr-lambda1和PCI-dhfr-SP163-lambda1。然后在 CHO(dhfr-)细胞中表达干扰素 lambda1:方法:使用 PCR 方法引入限制性酶切位点,并通过融合 PCR 将增强子与干扰素 lambda1 结合。测序后,将 Lambda1 和 SP163-lambda1 插入 PCI-dhfr 中,形成表达载体 PCI-dhfr-lambda1 和 PCI-dhfr-SP163-lambda1。然后用脂质体转染法将表达载体转染到 CHO(dhfr-)细胞中,用间接免疫荧光和 Western Blot 检测干扰素 lambda1 蛋白。用细胞病理效应抑制法评估了干扰素 lambda1 的抗病毒活性:结果:成功构建了λ干扰素的真核表达载体,并能表达λ干扰素1。免疫荧光结果显示,增强子促进了干扰素λ1的表达。用 Western Blot 检测转染 48 小时后 CHO(dhfr-)细胞中的干扰素 lambda1。细胞病理效应抑制表明表达的干扰素 lambda1 具有抗病毒活性:结论:在CHO(dhfr-)细胞中成功表达了λ1干扰素,且该蛋白具有抗病毒活性,这为建立稳定的λ1干扰素细胞系提供了重要依据。
[The expression of interferon-lambda1 in CHO cell].
Objective: To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells.
Methods: Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1.
Results: Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity.
Conclusion: Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.