EXPRESSION OF RECOMBINANT PROTEINS IN MILK OF TRANSGENIC ANIMALS
EXPRESSION OF RECOMBINANT PROTEINS IN MILK OF TRANSGENIC ANIMALS
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DOI：
10.1016/s1140-4639(05)80168-6
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发表时间：
1993-01-01
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影响因子：
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通讯作者：
HOUDEBINE, LM
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作者：
HOUDEBINE, LM
重组蛋白的大量生产可通过原核细胞或真核细胞实现。当蛋白质必须在转录后进行修饰（糖基化、磷酸化、切割、折叠等）时，可能需要高等真核生物的细胞。培养的高等脊椎动物细胞被用于制备诸如人凝血因子VIII和促红细胞生成素等蛋白质。有人提出利用转基因生物来达到同样的目的。实际上，一个完整的生物体能够实现非常强大的扩增，参与重组蛋白生物合成的细胞数量众多且处于最佳代谢条件下。生物体液（血液、乳汁、昆虫血淋巴、蛋清等）以及可能的转基因动物器官从理论上讲是重组蛋白的最佳来源。血液丰富，是屠宰场的副产品。其成分相对复杂，循环中的重组蛋白可能会严重影响动物的健康。乳汁非常丰富，其成分相对简单，蛋白酶含量低且易于收集。昆虫的血淋巴相对稀缺。当鸟类的转基因技术更容易实现时，蛋清将可能成为重组蛋白的一个来源。只有当重组蛋白的生物合成需要特定细胞类型时，才应考虑使用转基因动物的器官。因此，目前乳汁似乎是转基因动物重组蛋白的最佳来源。
大约有15个公立和私立实验室试图利用这些技术。这些技术包括制备含有一种乳汁蛋白基因调控区域以及待生产的相应蛋白质编码部分（cDNA或基因）的载体。将这些基因构建体转移到小鼠、兔子、绵羊、山羊、猪身上，表明这些技术确实很有前景。一种在转基因绵羊乳汁中生产的单一蛋白质——人α1 - 抗胰蛋白酶，目前已经达到了工业化生产规模。
这种方法有两个理论上的局限性：1）乳汁中分泌的一些蛋白质可能不像其天然形式那样成熟。到目前为止进行的实验（大约20种蛋白质已在实验规模上生产）表明乳腺细胞能够以正确的方式实现糖基化；2）相当比例的重组蛋白从乳腺的腺泡腔迁移到血液循环中，它们会影响哺乳期动物的健康。此外，这种方法仍然存在技术局限性，这些局限性在不久的将来可能会得到缓解：转基因表达控制不佳，往往不限于乳腺，含有cDNA而非基因的载体表达量低，获得大型转基因动物相对困难。
根据要制备的重组蛋白的量，可以使用几种动物。通过收集哺乳期动物离体乳腺在低温下自发分泌的乳汁，转基因小鼠可提供100毫克 - 1克的重组蛋白（用于结构和药物研究）。转基因兔子每年必须能从用挤奶机收集的乳汁中制备1千克的重组蛋白。对于更高量的蛋白质，山羊、绵羊、猪和牛当然是最佳候选动物。
如果这种方法的可行性不再需要证明，那么在工业规模上评估其实际成本仍然很困难。这当然是由于只有少数项目得以完成，而且该方法的技术细节仍在不断改进。一家美国公司所做的分析预测，到本世纪末，10%的药用重组蛋白将来自转基因动物的乳汁。这相当于一个2500万美元的年市场规模。
The bulky production of recombinant proteins can be achieved by procaryotes or eucaryotes cells. Cells from higher eucaryotes may be required when proteins have to be modified post-transcriptionnally (glycosylation phosphorylation, cleavage, folding...). Cells from higher vertebrates in culture are used to prepare proteins like human factor VIII and erythropoietin. The use of transgenic organism has been suggested to reach the same goal. Indeed a whole living organism allows a very potent amplification, the number of cells involved in the biosynthesis of the recombinant proteins being very numerous and in the best metabolic conditions.Biological fluids (blood, milk, insect hemolymph, egg white...) and possibly organs from transgenic animals are a priori the best sources of recombinant proteins. Blood is abundant and it is a by-product of slaughter house. Its composition is relatively complex and the circulating recombinant proteins may heavily alter health of animals. Milk is very abundant, its composition is relatively simple, it is poor in proteolytic enzymes and it can be collected easily. Hemolymph from insects is relatively scarce. Egg white will be a possible source of recombinant proteins, when transgenesis has become more accessible in birds. Organs from transgenic animals should be solicited only when a particular cell type is required for the biosynthesis of the recombinant proteins. Milk appears therefore, presently, as the best source of recombinant proteins from transgenic animals.About 15 public and private laboratories try to use these techniques. They consist in preparing vectors containing regulatory regions of one of the milk proteins genes and the coding part (cDNA or gene) of the corresponding proteins to be produced. The transfer of these gene constructs to mouse, rabbit, sheep, goat, pig, shows that these techniques are indeed very promising. A single protein, human alpha1-antitrypsin produced in milk of transgenic sheeps, has presently reached the preparation at an industrial scale. This method has two theoretical limitations: 1) some of the proteins secreted in milk may be not matured as their native counterparts. Experiments carried out so far (about 20 proteins has been produced at an experimental scale) indicate that the mammary cell is able to achieve glycosylation in a correct way; 2) a significant proportion of the recombinant proteins migrate from the alveolar compartment of the mammary gland to blood circulation and they can alter health of lactating animals. Moreover, this method has still technical limitations which will be likely alleviated in a near future : ill-controlled expression of the transgene which is often not restricted to the mammary gland, low expression with vectors containing cDNA rather than gene, relative difficulty to obtain large transgenic animals.According to the amount of recombinant proteins to be prepared, several animal species can be used. Transgenic mouse can provide 100 mg-1g of recombinant proteins (for structural and pharmaceutical studies) by collecting the milk which is spontaneously secreted from isolated mammary gland of lactating animals, incubated in cold. Transgenic rabbits must allow the preparation of 1 kg of recombinant proteins per year from their milk collected with a milking machine. For higher quantities of proteins, goat, sheep, pig and cow are of course the best candidates.If the feasibility of the method has no longer to be demonstrated, it remains uneasy to evaluate its real cost at an industrial scale. This is of course due to the fact that only a small number of projects have been led to term and that the technical details of the method is still permanently being improved. An analysis done by an american company predicts that, by the end of this century, 10% of the recombinant proteins for pharmaceutical use will come from milk of transgenic animals. This corresponds to an annual market of 25 millions dollars.