杆状病毒介导的哺乳动物细胞基因转移及其影响因素

杆状病毒作为优良的哺乳动物细胞基因转移载体已在基因治疗、疫苗开发、药物筛选以及基因调控研究等方面发挥巨大作用。但杆状病毒对哺乳动物细胞的嗜性不够广泛、转导效率和转基因表达水平偏低,以及表达持续时间短暂等问题制约其进一步发展,因此如何突破这个制约的瓶颈成为学者们新的关注焦点。针对以上问题,围绕杆状病毒介导哺乳动物细胞基因转移的各种影响因素进行综述。     

杆状病毒介导外源基因在哺乳动物细胞中表达的研究进展

杆状病毒(Baculovirus)是一种以昆虫为唯一宿主的病毒, 可用做生物杀虫剂或作为表达载体在昆虫细胞中大量表达外源蛋白, 制备疫苗。研究发现, 在哺乳动物细胞中携带哺乳动物启动子的重组杆状病毒能启动下游外源基因的表达但病毒不能在哺乳动物细胞中增值, 对细胞毒性小, 转导成功的细胞可以稳定传代并有效表达外源基因, 哺乳动物细胞比昆虫细胞对蛋白质具有更好的翻译后修饰, 表达出的蛋白结构更接近天然蛋白。因此, 杆状病毒可作为一种新型的哺乳动物细胞基因转移载体, 用于表达外源基因及作为一种基因治疗载体, 具有巨大潜力, 日益受到人们的关注。本文对杆状病毒作为一种表达载体在哺乳动物细胞中表达的研究进展进行了综述。     

杆状病毒介导对哺乳动物细胞的基因转移

将具有细胞毒效应的目的基因———Barnase基因置于CMV极早期启动子下游 ,利用Bac to Bac系统将上述表达盒插入AcNPV的ocu位相 ,构建重组病毒vAcCMVBar.感染HeLa等几种细胞后出现细胞毒性效应 ,其中一种人腹部肿瘤细胞的效应最为明显 ,剂量依赖分析是表达的Barnase所致 .因此认为 ,AcNPV是一种能感染具有一定的组织特异性的哺乳动物细胞的转基因载体 ,而Barnase基因表达产物造成细胞效应所需的量低于常规报告基因所需的评价水平     

杆状病毒载体及其在昆虫细胞中表达外源基因的研究进展

本文介绍了杆状病毒载体在昆虫细胞中表达外源基因的基本策略和发展趋势。杆状病毒载体系统近年来已被人们广泛用来表达人类、动物和植物等的一些重要蛋白质分子,在医学、农业等领域的基因工程研究中发挥了越来越大的作用。杆状病毒载体系统表达外源基因的效率高,表达产物的结构和活性与天然产物一致,为当今基因工程研究中最有发展前途的病毒载体表达系统。     

杆状病毒对不同哺乳动物细胞基因转移及表达效率的研究

研究已证实杆状病毒可进入某些哺乳动物细胞,这提示了可将重组杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。利用已构建的重组杆状病毒BacV-CMV-EGFPA,以含病毒的Sf9细胞培养上清同时孵育多种哺乳动物细胞,利用流式细胞术检测报告基因在不同细胞株中的转移效率及表达效率。共使用了20种哺乳动物细胞株,其中有12种人类组织细胞,7种小鼠组织细胞,1种猴组织细胞。实验结果显示携带CMV启动子的重组杆状病毒可有效进入多数哺乳动物细胞,其中对人、猴来源细胞的基因转移效率显著高于对鼠源细胞,对贴壁细胞的基因转移效率显著高于对悬浮细胞。同时,通过脂质体LipofectAMINE将携带有CMV启动子和EGFP基因的哺乳动物细胞表达质粒pCDNA3-1-EGFP分别转染部分特别是被认为杆状病毒进入能力较低的细胞株,结果显示CMV启动子在这些细胞中均可有效引导EGFP基因的表达,因此认为携带了CMV启动子的重组杆状病毒对不同哺乳动物细胞基因转移效率能基本反映出杆状病毒对不同种哺乳动物细胞的进入能力。通过综合比较携带CMV启动子的杆状病毒对不同种属及组织来源细胞的基因转移及表达效率,可看出杆状病毒对灵长类动物贴壁细胞的基因转移及表达效果是较好的,而对小鼠来源的细胞及悬浮培养细胞却并不十分理想,这表明将重组杆状病毒作为一种对哺乳动物细胞的基因转移工具,仍有其局限性,不一定对所有的细胞都合适,在应用时应予以充分考虑。     

杆状病毒用于哺乳动物细胞快速高效表达外源基因的研究

现已发现杆状病毒可进入某些培养的哺乳动物细胞,这提示可将杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。对杆状病毒转移载体的改造及对哺乳动物细胞的基因转移方式进行了进一步的研究。以绿色荧光蛋白基因为报告基因,利用Bac-to-Bac系统构建了分别含有正向和反向CMV启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达,但表达效率较低。用重组杆状病毒感染后Sf9细胞的培养上清直接与HepG2细胞作用,以流式细胞术检测基因转移效率及荧光表达强度,发现这两种病毒在相同的感染复数下对HepG2细胞具有相似的基因转移及表达效率。同时,利用流式细胞术进一步研究了直接使用重组杆状病毒感染4d后Sf9细胞的培养上清对哺乳动物细胞进行基因转移的方法。通过对HepG2细胞的实验结果显示,将带毒Sf9细胞培养上清(1.2×10⁷PFU/mL)用哺乳动物细胞培养基1倍稀释后,37℃下孵育靶细胞12h(moi=50),可达到较高的基因转移及表达效率,同时不会对细胞造成明显损伤。将重组杆状病毒与脂质体和逆转录病毒这两种系统对HepG2及CV1细胞的基因转移效率进行了比较,结果发现在同样未经浓缩等特殊处理的条件下重组杆状病毒对这两种细胞的基因转移效率是最高的。因此可以认为,经过适当改造后的Bac-to-Bac重组杆状病毒系统可作为一种对哺乳动物细胞简便高效的基因转移表达载体。     

乙型肝炎病毒表面抗原基因在杆状病毒载体与昆虫细胞体系中的表达

根据杆状病毒A组代表种苜蓿Y纹夜蛾核多角体病毒(Autographa californica Nuclear Po-lyhedrosis Virus,简称AcNPV)的分子生物学资料,我们采用分子剪裁和拼接手段,对AcNPV多角体蛋白基因加以修饰,并用人工合成定位探针,获得大约在该基因的ATG转译起始密码子一9处加有合成BamHl连接序列的转移载体质粒pAc—MV。 再与从乙肝病毒adw亚型的表面抗原基因HBng亚克隆株pYPss一1分出带有Ban·Hl粘性末端的HBsAg基因,构建表达载体质粒pAc—MV—HBsAgo经与野生型AcNPV DNA对Spodoptem 7rugJPerda细胞共转染,借助同源交换,获得插有HBsAg基因的多角体缺陷的重组病毒。根据HBsAg诊断血球凝集试验和HBs^g诊断酶标免疫测定,表明重组病毒使HBsAg基因在昆虫细胞中得到了表达,免疫电镜显示表达产物呈球状颗粒,大小约为22nm。表达产物粗制品按1μg／鼠对bal b／c鼠免疫,并于三周后强化免疫能产生抗体。由HBsAg纯样品酶标免疫法标定的标准曲线估计每升培养物的表达产物约{一8mg,细胞量为1一2×106个/ml.用重组病毒感染玉米螟4龄幼虫,也获得HBsAg基因的表达,展示了简易生产HB sag诊断试剂和疫苗的可喜前景。     

Recombinant baculoviruses as mammalian cell gene-delivery vectors

The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, recombinant baculovirus vectors engineered to contain mammalian cell-active promoter elements, have been used successfully for transient and stable gene delivery in a broad spectrum of primary and established mammalian cells. The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initiating a replication cycle and producing infectious virus in mammalian cells. The viruses can be readily manipulated, accommodate large insertions of foreign DNA, initiate little to no microscopically observable cytopathic effect in mammalian cells and have a good biosafety profile. These attributes will undoubtedly lead to the increased application and continued development of this system for efficient gene delivery into mammalian cells. Who said you can't teach an old dog new tricks?     

Recombinant baculoviruses as expression vectors for insect and mammalian cells.

Baculovirus expression vectors are widely used for expressing heterologous proteins in cultured insect cells. Recent advances include further development of the system for production of multi-subunit protein complexes, co-expression of protein-modifying enzymes to improve heterologous protein production, and additional applications of baculovirus display technology. The application of modified baculovirus vectors for gene expression in mammalian cells continues to expand.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

Multigene delivery in mammalian cells: Recent advances and applications

Systems for multigene delivery in mammalian cells, particularly in the context of genome engineering, have gained a lot of attention in biomolecular research and medicine. Initially these methods were based on RNA polymerase II promoters and were used for the production of protein complexes and for applications in cell biology such as reprogramming of somatic cells to stem cells. Emerging technologies such as CRISPR/Cas9-based genome engineering, which enable any alteration at the genomic level of an organism, require additional elements including U6-driven expression cassettes for RNA expression and homology constructs for designed genome modifications. For these applications, systems with high DNA capacity, flexibility and transfer rates are needed. In this article, we briefly give an update on some of recent strategies that facilitate multigene assembly and delivery into mammalian cells. Also, we review applications in various fields of biology that rely on multigene delivery systems.     

Rapid lineage-specific diversification of the mast cell chymase locus during mammalian evolution

Serine proteases constitute the major protein granule content of cells of several hematopoietic cell lineages. A subgroup of these proteases, including the mast cell chymases, neutrophil cathepsin G, and T cell granzymes B to F and N, are in all investigated mammals encoded in one locus, the chymase locus. It is interesting to note that this locus has diversified greatly during the last 95 Myr of mammalian evolution. This divergence is exemplified by the presence of Mcpt8-related genes and multiple β-chymases in the mouse and rat, which lack direct counterparts in primates and in seven functional granzyme genes in the mouse where the human locus has only two. To study the expansion of the locus during rodent evolution and to better understand the evolutionary origin of β-chymases and the Mcpt8-family, we have performed a detailed analysis of the chymase locus of four mammalian species, i.e., human, dog, mouse, and rat. As a result, we report here a second chymase-like gene in dog, Cma2, which clusters with β-chymases in phylogenetic analyses. This finding supports a duplication of the common ancestor for α- and β-chymases before the major radiation of placental mammals, and a loss of the ancestral β-chymase gene sometime during primate evolution. Moreover, we show that in the rat, the Mcpt8-family diversified relatively recently together with sequences related to the β-chymase Mcpt2. Eight novel genes were identified in the duplication region, four of which are predicted to be functional. Duplications of rat granzyme B- and C-like sequences occurred seemingly independently within a similar time frame, but did not give rise to functional genes. Due to the duplications in rat and deletions in the carnivore/primate lineage, the rat chymase locus is approximately 15 and 9 times larger than its counterparts in dog and human, respectively. These findings illustrate the importance of gene duplications in conferring rapid changes in mammalian genomes.     

Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process

Various mechanistic and black-box models were applied for on-line estimations of viable cell concentrations in fed-batch cultivation processes for CHO cells. Data from six fed-batch cultivation experiments were used to identify the underlying models and further six independent data sets were used to determine the performance of the estimators. The performances were quantified by means of the root mean square error (RMSE) between the estimates and the corresponding off-line measured validation data sets. It is shown that even simple techniques based on empirical and linear model approaches provide a fairly good on-line estimation performance. Best results with respect to the validation data sets were obtained with hybrid models, multivariate linear regression technique and support vector regression. Hybrid models provide additional important information about the specific cellular growth rates during the cultivation.     

Design and synthesis of a Magainin2 fusion protein gene suitable for a mammalian expression system

We have designed and synthesized a gene encoding a fusion protein comprising Magainin2 and a carrier protein with the aim of screening for a suitable carrier protein expressing antibacterial peptides in the mammalian expression system. The antibacterial peptide Magainin2 was used as a model. Our results on mammalian cell expression showed that there was no exceptional splicing in the transfected CHO-s cells. Analysis of the transgenic mouse model revealed that the expression level of this fusion protein in one transgenic positive mouse was up to 10 g/l, which is close to the level of beta-casein in goat milk. The bioactivity analysis showed that the digested fusion protein had antibacterial activity. These results demonstrate that the synthetic gene of the carrier protein is suitable for expressing an antibacterial peptide in a mammalian cell system at high productivity and efficiency. Moreover, they demonstrate the potential for producing antibacterial peptides in a transgenic animal bioreactor on a large scale and inexpensively.     

Lipophosphonate/lipophosphoramidates: a family of synthetic vectors efficient for gene delivery

Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).     

人绒毛膜促性腺激素β－亚基在中国仓鼠卵巢细胞中的表达

本文以哺乳动物细胞表达载体ｐＳＶ２－ｄｈｆｒ为运载体，构建了受控于ＳＶ４０早期启动子β－ｈＣＧ基因的表达载体，转染ＣＨＯ－ｄｈｆｒ－细胞后，挑选ＣＨＯ（ｄｈｆｒ－）细胞，结果表明β－ｈＣＧ不仅在细胞中表达，还分泌到培养液中。通过氨甲喋呤（ＭＴＸ）加压后，表达量逐渐提高，０．１μｍｏｌ／ＬＭＴＸ条件下培养液中β－ｈＣＧ最高表达量可达到１．５μｇ／１０６细胞·２４小时。经亲和层析获得纯的重组β－ｈＣＧ，其分子量与天然制品一致，放射免疫测定的免疫原性为天然制品的８４．９％。