人乳过氧化物酶（Lactoperoxidase）转基因小鼠模型的建立
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摘要
乳腺生物反应器是一种利用动物转基因技术在乳腺细胞中表达多肽药物、工业酶、疫苗和抗体等蛋白的技术。该技术具有低投入高产出的特点，其效率是利用以大肠杆菌和动物细胞培养技术的一百倍，是一种非常有潜力的高新技术。1987年，Simons等人在转基因小鼠乳腺中首次成功地表达羊乳球蛋白基因，并且小鼠奶样中的蛋白含量高达23克／升，大约是动物细胞表达蛋白的400倍以上。该技术一经出现就得到迅猛的发展。许许多多的著名科学家都认为这将是畜牧业前所未有的革命，可能会给社会带来巨大的经济效益。尤其，随着动物克隆技术成熟，转基因技术得到了更充分的发展。这将使乳腺生物反应器的大规模生产更加现实，因此一场围绕该技术的国际性竞争将是必然。
本项研究主要是建立在乳腺细胞中高效表达人乳过氧化物酶(LPO)基因的转基因小鼠模型。人乳过氧化物酶属于哺乳动物亚铁血红素超家族，广泛存在于各种哺乳动物乳汁及其它分泌物中，在过氧化氢存在的情况下可以氧化硫氰酸和其它一些物质，通过这种反应乳过氧化物酶就可以杀死或抑制细菌，同时清除氧自由基。因此它除了可以抑杀细菌、预防疾病外又是一种延缓衰老的天然保健品，是哺乳动物防卫系统中重要的组成部分。它的价值已经越来越为人们所重视，现今已经在乳制品的保藏、饲料添加、临床医学上得到广泛应用。
人乳中乳过氧化物酶占人乳中所有蛋白总量的0.004％，活性约为牛乳的1／20。随着对奶制品需求的增加，奶制品的品质也越来越受到人们的关注。利用乳腺生物反应器的方法改造奶牛、奶山羊，使生产出的奶成分近似于人奶，既有营养的功能，又有药用的功能。这种奶可以使孩子长得高大，促进大脑和神经发育，增强免疫功能，这种新型保健品一定会有广阔的前途。我们的研究就是以小鼠为动物模型进行乳腺特异表达外源人乳过氧化物酶酶的研究。
为了获得乳腺高效表达人乳过氧化物酶的小鼠模型，我们构建了pBC1-hLPO人乳过氧化物酶基因表达载体，核期小鼠胚胎制备转基因小鼠．
载体pBC1-hLPO由山羊beta-casein启动子指导，还包含鸡bata-globin隔离子、及外显子1、2和内含子1。利用显微注射方法注射原共出生28只，用PCR和Southern杂交的方法检测在出生5只为转基因小鼠，转基因阳性率约为18％。
为检测转基因小鼠的乳样中人乳过氧化物酶的表达情况，根据人乳过氧化物酶的三级预测结构，设计合成多肽CSAID KLDLSPWASV KN，与KLH偶联后免疫大白兔，取抗血清用ELISA法检测，效价达500万倍，完全可以用于转基因小鼠乳样的Western检测。
The mammary gland bioreactor is the technique that a kind of using animal transgenic technology to express proteins, in mammalian cells, such as pharmaceutical polypeptide, industry enzyme , vaccine, antibody and etc. This potential technique is characterized by producing high output in low cost, its efficiency is as 100 times as that of cultivating echerichia coli and animal cells. In 1987, the Simons first successfully expressed sheep milk globulin gene in mammary glands of transgenic mice. What' s more, the concentation of this recombinant protein is up to 23 g/1, which is about 400 times higher than that expressed by animal cells. The technique advanced with remarkable speed since its appear. Many famous scientists all consider it is an unprecedented revolution in animal husbandry which may bring the society enormous economic results. Especially, with the development of clone, the transgenic technology also has got well developed. This will make the large-scale production of the mammary glands bioreactor
more realistic, therefore an international competition around this technique will be inevitable.
This research is to establish a model of transgenic mice which can express human lactoperoxidase (LPO)efficiently in mammary gland cells mainly. Lactoperoxidase belongs to mammalian heme peroxidase superfamily , it can widely be found in milk and other secretory fluid . It can oxidate thiocyanate and othe substrates under the circumstances of H202, by this reaction, human lactoperoxidase can kill or inhibit the germs , and eliminate H-A at the same time. In consequence.it is also a natural health care goods which can delay slow death in addition to preventing disease by repressing and killing bacterium and is an essential component of the defence system in mammalian.Its value is more and more valueed by people , Nowadays, it has been widely used in keeping in store of dairy products fodder increasing , and on the clinical medicine
actoperoxidase whose activity is correspond to 1/20 of cows represents about 0. 004% of the total protein in crude human milk. With increase in milk consumptions, the quality of milk is more concerned. It is through mammary gland bioreactor that milk of bovine and sheep may become similar to human milk, which has nutritional and medical function. The reconstructive milk is a promising product, which promotes children growth and development of nerve system and brain and immunity ability. Our research is to establish transgenic mouse model which highly expresses recombinant human lactoperoxidase in mammary gland.
In order to establish the mouse model, pBCl-hLPO was constructed.
Transgenic mice were generated by fertilized oocyte microinjection with the constructs.
pBCl-hLPO expression vector is composed of Chicken beta-globin insulator, Goat beta-casein promoter, beta-casein exon K 2, beta-casein intron 1, the human lactoperoxidase encoding region and 3' termination region. As for pBOhLPO expression vector, 28 mice were born, and Spositive transgenic mice were identified by PCR and confirmed by Southern Blotting analysis of tail DNA. The integration rate is 17.86%. In order to examinate repression of human lactoperoxidase in the milk of transgenic mice ,based on the third class predict construction of human lactoperoxidase, Polypeptide CSAID KLDLSPWASV KN is synthetised in the design .united with KLH and then immunited white rabbits , examinated antibody got from the rabbits in the way of ELISA' s law , The effect price is up to 5 million times , that is to say , it may be used in Western blotting examinition the milk of transgenic mice completely
本项研究主要是建立在乳腺细胞中高效表达人乳过氧化物酶(LPO)基因的转基因小鼠模型。人乳过氧化物酶属于哺乳动物亚铁血红素超家族，广泛存在于各种哺乳动物乳汁及其它分泌物中，在过氧化氢存在的情况下可以氧化硫氰酸和其它一些物质，通过这种反应乳过氧化物酶就可以杀死或抑制细菌，同时清除氧自由基。因此它除了可以抑杀细菌、预防疾病外又是一种延缓衰老的天然保健品，是哺乳动物防卫系统中重要的组成部分。它的价值已经越来越为人们所重视，现今已经在乳制品的保藏、饲料添加、临床医学上得到广泛应用。
人乳中乳过氧化物酶占人乳中所有蛋白总量的0.004％，活性约为牛乳的1／20。随着对奶制品需求的增加，奶制品的品质也越来越受到人们的关注。利用乳腺生物反应器的方法改造奶牛、奶山羊，使生产出的奶成分近似于人奶，既有营养的功能，又有药用的功能。这种奶可以使孩子长得高大，促进大脑和神经发育，增强免疫功能，这种新型保健品一定会有广阔的前途。我们的研究就是以小鼠为动物模型进行乳腺特异表达外源人乳过氧化物酶酶的研究。
为了获得乳腺高效表达人乳过氧化物酶的小鼠模型，我们构建了pBC1-hLPO人乳过氧化物酶基因表达载体，核期小鼠胚胎制备转基因小鼠．
载体pBC1-hLPO由山羊beta-casein启动子指导，还包含鸡bata-globin隔离子、及外显子1、2和内含子1。利用显微注射方法注射原共出生28只，用PCR和Southern杂交的方法检测在出生5只为转基因小鼠，转基因阳性率约为18％。
为检测转基因小鼠的乳样中人乳过氧化物酶的表达情况，根据人乳过氧化物酶的三级预测结构，设计合成多肽CSAID KLDLSPWASV KN，与KLH偶联后免疫大白兔，取抗血清用ELISA法检测，效价达500万倍，完全可以用于转基因小鼠乳样的Western检测。
The mammary gland bioreactor is the technique that a kind of using animal transgenic technology to express proteins, in mammalian cells, such as pharmaceutical polypeptide, industry enzyme , vaccine, antibody and etc. This potential technique is characterized by producing high output in low cost, its efficiency is as 100 times as that of cultivating echerichia coli and animal cells. In 1987, the Simons first successfully expressed sheep milk globulin gene in mammary glands of transgenic mice. What' s more, the concentation of this recombinant protein is up to 23 g/1, which is about 400 times higher than that expressed by animal cells. The technique advanced with remarkable speed since its appear. Many famous scientists all consider it is an unprecedented revolution in animal husbandry which may bring the society enormous economic results. Especially, with the development of clone, the transgenic technology also has got well developed. This will make the large-scale production of the mammary glands bioreactor
more realistic, therefore an international competition around this technique will be inevitable.
This research is to establish a model of transgenic mice which can express human lactoperoxidase (LPO)efficiently in mammary gland cells mainly. Lactoperoxidase belongs to mammalian heme peroxidase superfamily , it can widely be found in milk and other secretory fluid . It can oxidate thiocyanate and othe substrates under the circumstances of H202, by this reaction, human lactoperoxidase can kill or inhibit the germs , and eliminate H-A at the same time. In consequence.it is also a natural health care goods which can delay slow death in addition to preventing disease by repressing and killing bacterium and is an essential component of the defence system in mammalian.Its value is more and more valueed by people , Nowadays, it has been widely used in keeping in store of dairy products fodder increasing , and on the clinical medicine
actoperoxidase whose activity is correspond to 1/20 of cows represents about 0. 004% of the total protein in crude human milk. With increase in milk consumptions, the quality of milk is more concerned. It is through mammary gland bioreactor that milk of bovine and sheep may become similar to human milk, which has nutritional and medical function. The reconstructive milk is a promising product, which promotes children growth and development of nerve system and brain and immunity ability. Our research is to establish transgenic mouse model which highly expresses recombinant human lactoperoxidase in mammary gland.
In order to establish the mouse model, pBCl-hLPO was constructed.
Transgenic mice were generated by fertilized oocyte microinjection with the constructs.
pBCl-hLPO expression vector is composed of Chicken beta-globin insulator, Goat beta-casein promoter, beta-casein exon K 2, beta-casein intron 1, the human lactoperoxidase encoding region and 3' termination region. As for pBOhLPO expression vector, 28 mice were born, and Spositive transgenic mice were identified by PCR and confirmed by Southern Blotting analysis of tail DNA. The integration rate is 17.86%. In order to examinate repression of human lactoperoxidase in the milk of transgenic mice ,based on the third class predict construction of human lactoperoxidase, Polypeptide CSAID KLDLSPWASV KN is synthetised in the design .united with KLH and then immunited white rabbits , examinated antibody got from the rabbits in the way of ELISA' s law , The effect price is up to 5 million times , that is to say , it may be used in Western blotting examinition the milk of transgenic mice completely
引文
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