Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.
Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.
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DOI：
10.1186/1742-4690-11-33
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发表时间：
2014-04-25
期刊：
影响因子：
3.3
通讯作者：
Cupo A
中科院分区：
文献类型：
--
作者：
Chung NP;Matthews K;Kim HJ;Ketas TJ;Golabek M;de Los Reyes K;Korzun J;Yasmeen A;Sanders RW;Klasse PJ;Wilson IA;Ward AB;Marozsan AJ;Moore JP;Cupo A
基于A亚型BG 505序列的重组可溶性切割HIV-1包膜糖蛋白SOSIP.664 gp 140三聚体正在进行结构研究，并在动物中作为免疫原进行测试。为了使这些三聚体成为人体试验的候选疫苗，它们需要以可接受的质量以适当的量制备。通过瞬时转染完成这样的任务可能是具有挑战性的。大量表达重组蛋白的传统方法是通过永久细胞系，通常是哺乳动物来源的。制备产生BG 505 SOSIP.664三聚体的细胞系需要共表达弗林蛋白酶，以确保gp 120和gp 41亚基之间的切割位点得到充分利用。我们设计了能够表达Env和Furin的载体，并使用它通过位点特异性重组来创建Stable 293 T和CHO Flp-In™细胞系。这两种细胞系都能产生高质量的裂解三聚体，产量高达12-15 mg/1 × 109个细胞。在初始接种后，这种水平的三聚体表达维持长达30天（10次传代），并且始终上级通过瞬时转染可以实现的表达。电子显微镜研究证实，纯化的三聚体具有相同的天然样的外观，通过瞬时转染和用于产生高分辨率的结构。它们还具有适当的抗原特性，包括呈递广泛中和抗体PGT 145的四级表位。表达BG 505 SOSIP.664三聚体的细胞系可产生适当质量的蛋白质，用于结构研究和动物免疫原性实验。该方法适用于在药品生产质量管理规范条件下制备类似的生产线，以生产用于人体临床试验的三聚体。此外，任何env基因都可以掺入该载体系统中，从而允许通过瞬时转染或从稳定细胞系从多种基因型制造SOSIP三聚体。
Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12–15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.
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影响因子：
64.8
作者：
Liu, Jun;Bartesaghi, Alberto;Borgnia, Mario J.;Sapiro, Guillermo;Subramaniam, Sriram
通讯作者：
Subramaniam, Sriram
影响因子：
6.7
作者：
Sanders RW;Derking R;Cupo A;Julien JP;Yasmeen A;de Val N;Kim HJ;Blattner C;de la Peña AT;Korzun J;Golabek M;de Los Reyes K;Ketas TJ;van Gils MJ;King CR;Wilson IA;Ward AB;Klasse PJ;Moore JP
通讯作者：
Moore JP
影响因子：
5.4
作者：
Binley, JM;Sanders, RW;Moore, JP
通讯作者：
Moore, JP
影响因子：
1.6
作者：
Kirschner, Marc;Monrose, Val;Moore, John P.
通讯作者：
Moore, John P.
影响因子：
4.8
作者：
Depetris, Rafael S.;Julien, Jean-Philippe;Moore, John P.
通讯作者：
Moore, John P.