人血小板生成素乳腺表达载体的构建
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摘要
乳腺生物反应器的构建关键在选择适宜的启动子和目的基因,才能保证目的基因在动物乳腺细胞中的高效、特异性表达,根据这要求选用的启动子一般是动物乳蛋白基因。酪蛋白是哺乳动物乳汁中主要的蛋白质,广泛地存在于反刍类和啮齿类动物的乳汁中,其中β-酪蛋白是一种高效表达的乳蛋白,是乳汁中含量较高的乳蛋白之一。在小鼠乳汁约占总蛋白的20%,在奶牛乳汁约占总蛋白的27%,在其它哺乳动物的乳汁中也占20～30%。在泌乳激素的刺激下,β-酪蛋白具有很强的表达活性,因此其调控成分能够指导靶基因在乳腺组织中高效表达。
为了构建动物乳腺表达载体,分别以荷斯坦奶牛β-酪蛋白和近交系C57/B6小鼠β-酪蛋白基因作为启动子;以中国人血小板生成素作为目的基因。将牛β-酪蛋白、小鼠β-酪蛋白和人血小板生成素与T载体(pMD-19载体)连接,用α互补蓝-白斑筛选法进行阳性筛选,将阳性菌液测序结果表明其同源性与NCBI里公布的序列达94%以上,保证下一步试验操作的准确性。利用限制内切酶将牛β-酪蛋白、小鼠β-酪蛋白和人血小板生成素从T载体切出,利用琼脂糖凝胶回收试剂盒回收、纯化;酶切载体pcDNA3.1(+)与之有相同的酶切位点,然后利用T4 DNA连接酶连接,用PCR和酶切鉴定,最终将启动子(牛β-酪蛋白和小鼠β-酪蛋白)、目的基因(人血小板生成素)与载体pcDNA3.1(+)连接,构建高效、特异性表达载体,为下一步目的基因的表达提供准备条件。试验结果如下:
1.克隆了约7.8Kb牛β-酪蛋白和约4.8Kb小鼠β-酪蛋白,分别作为启动子;克隆了约6.1Kb人血小板生成素,作为目的基因构建乳腺特异表达载体。
2.测序结果表明,克隆序列与NCBI里公布的序列其同源性在94%以上,可以进行下一步试验操作。
3.利用限制内切酶将目的基因从T载体中切出,用T4 DNA连接酶将目的基因与载体pcDNA3.1(+)连接,以备构建特异性表达载体。
Construction of mammary gland bioreactor is the key to choose suitable promoter and foreign gene, which is the milk protein gene, the foreign gene can be high-effective, specific expression in breast cells. Casein is the main protein in the milk of mammals, widely existed in ruminant and rodents animals. Theβ-casein is one of high-effective expression and contention in the milk protein. In the milk protein of mice and cow respectively contented about 20%, 27%,and 20～30% in other mammals. In the stimulation of galactin, theβ-casein has strongly express-activeness, which regulation element can guide the target gene high-efficiency expression in breast tissue.
In order to construction of mammary gland expression-vector, we respectively obtained the bovineβ-casein (bβ-casein)of Holstein cow , mouseβ-casein (mβ-casein)of C57/B6 mice as the promoter, human Thrombopoietin(hTPO) of Chinese as foreign gene . We linked the bβ-casein, mβ-casein and hTPO gene with T- vector (pMD-19 vector), respectively .Then we used theαcomplementary action blue - white spot filtration method filtrated positive cloning.The result of the positive bacterium liquid sequencing showed that the identity of genome sequencing was over 94% which contrasted with the published in NCBI, it can be carry out the next experiment.We cut the bβ-casein, mβ-casein and hTPO gene from the T- vector, which used the restriction endonucl- ease digestion, then we reclaimed and sublimated it using the Agarose Gel Purification Kit. pcDNA3.1 (+) vector had the same restriction endonuclease site to bβ-casein, mβ-casein and hTPO gene ,and then linked it with them using T4 DNA ligase and we identificated it using PCR and restricttion endonuclease digestion, finally we will be link promoter (bβ-casein, mβ-casein) , the foreign gene(hTPO) with the pcDNA3.1 (+)vector, constructed the high-effective, specific expression-vector to provide condition for the expression of the target gene . We got the results as follow:
1. As promoter we cloned about 7.8Kb bβ-casein and about 4.8 Kb mβ-casein gene, respectively. As foreign gene we cloned about 6.1Kb hTPO.
2. The result of sequencing showed that the identity of genome sequencing was over 94 % which contrasted with the published in NCBI, it can be carry out the next experiment.
3. We cut target gene from the T-vector using restriction endonuclease digestion, and then linkes the target gene with the pcDNA3.1 (+) vector using T4 DNA ligase to construction of specific expression-vector.
为了构建动物乳腺表达载体,分别以荷斯坦奶牛β-酪蛋白和近交系C57/B6小鼠β-酪蛋白基因作为启动子;以中国人血小板生成素作为目的基因。将牛β-酪蛋白、小鼠β-酪蛋白和人血小板生成素与T载体(pMD-19载体)连接,用α互补蓝-白斑筛选法进行阳性筛选,将阳性菌液测序结果表明其同源性与NCBI里公布的序列达94%以上,保证下一步试验操作的准确性。利用限制内切酶将牛β-酪蛋白、小鼠β-酪蛋白和人血小板生成素从T载体切出,利用琼脂糖凝胶回收试剂盒回收、纯化;酶切载体pcDNA3.1(+)与之有相同的酶切位点,然后利用T4 DNA连接酶连接,用PCR和酶切鉴定,最终将启动子(牛β-酪蛋白和小鼠β-酪蛋白)、目的基因(人血小板生成素)与载体pcDNA3.1(+)连接,构建高效、特异性表达载体,为下一步目的基因的表达提供准备条件。试验结果如下:
1.克隆了约7.8Kb牛β-酪蛋白和约4.8Kb小鼠β-酪蛋白,分别作为启动子;克隆了约6.1Kb人血小板生成素,作为目的基因构建乳腺特异表达载体。
2.测序结果表明,克隆序列与NCBI里公布的序列其同源性在94%以上,可以进行下一步试验操作。
3.利用限制内切酶将目的基因从T载体中切出,用T4 DNA连接酶将目的基因与载体pcDNA3.1(+)连接,以备构建特异性表达载体。
Construction of mammary gland bioreactor is the key to choose suitable promoter and foreign gene, which is the milk protein gene, the foreign gene can be high-effective, specific expression in breast cells. Casein is the main protein in the milk of mammals, widely existed in ruminant and rodents animals. Theβ-casein is one of high-effective expression and contention in the milk protein. In the milk protein of mice and cow respectively contented about 20%, 27%,and 20～30% in other mammals. In the stimulation of galactin, theβ-casein has strongly express-activeness, which regulation element can guide the target gene high-efficiency expression in breast tissue.
In order to construction of mammary gland expression-vector, we respectively obtained the bovineβ-casein (bβ-casein)of Holstein cow , mouseβ-casein (mβ-casein)of C57/B6 mice as the promoter, human Thrombopoietin(hTPO) of Chinese as foreign gene . We linked the bβ-casein, mβ-casein and hTPO gene with T- vector (pMD-19 vector), respectively .Then we used theαcomplementary action blue - white spot filtration method filtrated positive cloning.The result of the positive bacterium liquid sequencing showed that the identity of genome sequencing was over 94% which contrasted with the published in NCBI, it can be carry out the next experiment.We cut the bβ-casein, mβ-casein and hTPO gene from the T- vector, which used the restriction endonucl- ease digestion, then we reclaimed and sublimated it using the Agarose Gel Purification Kit. pcDNA3.1 (+) vector had the same restriction endonuclease site to bβ-casein, mβ-casein and hTPO gene ,and then linked it with them using T4 DNA ligase and we identificated it using PCR and restricttion endonuclease digestion, finally we will be link promoter (bβ-casein, mβ-casein) , the foreign gene(hTPO) with the pcDNA3.1 (+)vector, constructed the high-effective, specific expression-vector to provide condition for the expression of the target gene . We got the results as follow:
1. As promoter we cloned about 7.8Kb bβ-casein and about 4.8 Kb mβ-casein gene, respectively. As foreign gene we cloned about 6.1Kb hTPO.
2. The result of sequencing showed that the identity of genome sequencing was over 94 % which contrasted with the published in NCBI, it can be carry out the next experiment.
3. We cut target gene from the T-vector using restriction endonuclease digestion, and then linkes the target gene with the pcDNA3.1 (+) vector using T4 DNA ligase to construction of specific expression-vector.
引文
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