The production of cleaved, trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent
The production of cleaved, trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent
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DOI：
10.1016/j.pep.2006.02.017
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发表时间：
2006-07-01
影响因子：
1.6
通讯作者：
Moore, John P.
中科院分区：
文献类型：
--
作者：
Kirschner, Marc;Monrose, Val;Moore, John P.
三聚体HIV-1包膜糖蛋白（Env）代替单体gp 120作为疫苗抗原，因为它们更接近地模拟病毒体表面上表达的刺突。因此，可以认为三聚体具有比gp 120更天然的结构，因此在产生中和抗体方面可能是上级的。制备Env三聚体的一种方法是确保它们在gp 120 gp 41边界处被切割，但通过其他工程化取代如亚基内二硫键（SOS和SOSIP gp 140蛋白）来稳定。然而，正确折叠、切割的三聚体的生产由于需要共表达外源蛋白酶弗林蛋白酶以促进gp 120-gp 41切割位点的有效加工而变得复杂。此外，纯化的三聚体SOSIP gp 140蛋白的产率通常是中等的，并且对于放大程序，转染试剂的成本成为重要的经济因素。在这里，我们评估了这些复杂蛋白质瞬时表达的最佳培养条件。我们发现，使用线性聚乙烯亚胺25 kDa（PEI 25 k）作为转染助剂是一种具有成本效益的。是几种市售产品的经济替代品。通过使用PEI 25 k和优化的质粒：弗林蛋白酶比率，我们可以表达蛋白水解成熟的三聚体Env疫苗抗原，其水平足够高以用于免疫或结构研究。我们还表明，相同的转染方法可用于产生感染性假病毒。(c)2006年爱思唯尔公司All rights reserved.
Trimeric HIV-1 envelope glycoproteins (Env) are now being evaluated instead of monomeric gp120 as vaccine antigens because they mimic more closely the spikes expressed on the surface of virions. Thus, it can be argued that trimers have a more native structure than gp120, so might be superior at raising neutralizing antibodies. One approach to making Env trimers is to ensure that they are cleaved at the gp120 gp41 border, but stabilized by other, engineered substitutions such as intra-subunit disulfide bonds (SOS and SOSIP gp140 proteins). However, the production of properly folded, cleaved trimers is complicated by the requirement for co-expression of the exogenous protease furin, to facilitate the efficient processing oft the gp120-gp41 cleavage site. Also, yields of purified trimeric SOSIP gp140 proteins are usually moderate and for scale-up procedures the cost of transfection reagents becomes an important economical factor. Here, we assess the optimal culture conditions for the transient expression of these complex proteins. We found that the use of linear polyethylenimine 25 kDa (PEI25k) as a transfection aid was a cost-efficient. economical alternative to several commercially available products. By using PEI25k and an optimized plasmid:furin ratio, we could express proteolytically mature, trimeric Env vaccine antigens at levels high enough for use in immunization or structural studies. We also show that the same transfection method can be used to generate infectious pseudoviruses. (c) 2006 Elsevier Inc. All rights reserved.