牛αs1-casein乳腺表达载体及金属硫蛋白在转基因小鼠乳腺中的表达
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摘要
金属硫蛋白是一类富含半胱氨酸、无芳香族氨基酸的低分子量蛋白，可分为四种亚型MT-I、MT-II、MT-III、MT-IV，每分子MT可以结合7分子的Zn2+或Cd2+。对金属离子与自由基具有很高亲和力的特性使得MT在对金属离子特别是Zn2+的代谢、运输以及自由基的清除过程中发挥着重要作用，因此在商业和医学领域有着广泛的应用，但传统制备方法效率低、成本高的局限性限制了MT的大规模应用，因此我们借助乳腺生物反应器技术以解决这种问题。
首先，利用牛αs1酪蛋白的两侧序列-1660-1437,15628-19476作为MT的调控元件，1.3kb鸡溶菌酶基因5’侧翼序列和2个拷贝的鸡β珠蛋白绝缘子作为去除位置效应的原件，三者通过组合得到三种结构的表达载体pIMC-MT、pIC-MT、pMC-MT。
其次，采用限制性内切酶切割三个表达载体，并将得到的包含结构元件的片段通过原核显微注射技术获得转基因鼠，在F0代鼠中经PCR和Southern Blot鉴定pIMC-MT共有7只阳性鼠（公鼠3、14、16、20，母鼠22、24、29），pIC-MT共有7只阳性鼠（公鼠2、7、8、10、13，母鼠21、27），pMC-MT共有阳性鼠3只（公鼠6、12，母鼠20）。由于母鼠少，所以对阳性鼠传代以扩大阳性母鼠数量。经PCR鉴定F1代鼠中pIMC-MT共8只阳性母鼠（8、10、11、20、22、23、36、44），pIC-MT共3只阳性母鼠(6、10、28)，pMC-MT共1只阳性母鼠（25）。
最后，对F0代和F1代母鼠分别采集乳汁，并对F1代采集乳腺组织进行Western blot、RT-PCR及Real-time PCR鉴定。在蛋白水平上所有转基因鼠均未检测到MT的表达，只在RNA水平上检测到F1代小鼠中MT的表达，pIMC-MT转基因鼠中只有11号有表达，pIC-MT及pMC-MT转基因鼠中全部有表达。与GAPDH相比，GAPDH的表达量分别是pIMC-MT-F1-11的17.87、pIC-MT-F1-6的2.25、pIC-MT-F1-10的4.92、pIC-MT-F1-28的14.32倍，唯独pMC-MT-F1-25的倍数为GAPDH的14.93。
MT is a cysteine-rich, low molecular weight and non-aromatic amino acid protein,andhas a wide range of applications in the field of business and medicine. The traditionalpreparation method of MT has, however restricted its applications,we set out to resolve thepromble with mammary gland bioreactor technology.
First, we use the flanking sequence of the bovine αs1-casein gene,-1660-1437,15628-19476, as regulatory elements of MT. The5' flanking sequence of chickenlysozyme gene and two copies of the chicken β-globin protein insulators were used toeliminate position effects. Through the combination of the three structures, we obtained threeexpression vector pIMC-MT, pIC-MT, pMC-MT.
Secondly, the expression vectors were digested by restriction enzyme and thefragement contain the structure elements were used to generate transgenic mice by pronuclearinjection. F0generation mice were screened by PCR and Southern Blot. The pIMC-MT hadseven positive mice (male mice3,14,16,20, female mice22,24,29),and the pIC-MT hadseven positive mice (male mice2,7,8,10,13, female mice21,27),while pMC-MT had threepositive mice (male mice6,12, female mice20). In order to propagate the number of postivefamale mice, F0postive mice were mated to WT mice. Eight postive female mice wereobtained for PIMC-MT (8,10,11,20,22,23,36,44), three for pIC-MT (6,10,28), and onlyone for pMC-MT (25).
We finally, collected the milk from F0and F1generation, and breast tissue from F1generation. The expression of MT was examined by Western Blot, RT-PCR and Real-timePCR. We could not detect the expression of MT at the protein level. Transcription of MT wasdetected in F1generation of pIMC-MT-F1-11, and all mice of pIC-MT and pMC-MT.Compared to GAPDH, the level of MT transcription was17.87-fold less in pIMC-MT-F1-11,2.25-fold less in pIC-MT-F1-6,4.92-fold less in pIC-MT-F1-10,14.32-fold less inpIC-MT-F1-28. Only in pMC-MT-F1-25,MT was expressed25-fold higher than GAPDH.
首先，利用牛αs1酪蛋白的两侧序列-1660-1437,15628-19476作为MT的调控元件，1.3kb鸡溶菌酶基因5’侧翼序列和2个拷贝的鸡β珠蛋白绝缘子作为去除位置效应的原件，三者通过组合得到三种结构的表达载体pIMC-MT、pIC-MT、pMC-MT。
其次，采用限制性内切酶切割三个表达载体，并将得到的包含结构元件的片段通过原核显微注射技术获得转基因鼠，在F0代鼠中经PCR和Southern Blot鉴定pIMC-MT共有7只阳性鼠（公鼠3、14、16、20，母鼠22、24、29），pIC-MT共有7只阳性鼠（公鼠2、7、8、10、13，母鼠21、27），pMC-MT共有阳性鼠3只（公鼠6、12，母鼠20）。由于母鼠少，所以对阳性鼠传代以扩大阳性母鼠数量。经PCR鉴定F1代鼠中pIMC-MT共8只阳性母鼠（8、10、11、20、22、23、36、44），pIC-MT共3只阳性母鼠(6、10、28)，pMC-MT共1只阳性母鼠（25）。
最后，对F0代和F1代母鼠分别采集乳汁，并对F1代采集乳腺组织进行Western blot、RT-PCR及Real-time PCR鉴定。在蛋白水平上所有转基因鼠均未检测到MT的表达，只在RNA水平上检测到F1代小鼠中MT的表达，pIMC-MT转基因鼠中只有11号有表达，pIC-MT及pMC-MT转基因鼠中全部有表达。与GAPDH相比，GAPDH的表达量分别是pIMC-MT-F1-11的17.87、pIC-MT-F1-6的2.25、pIC-MT-F1-10的4.92、pIC-MT-F1-28的14.32倍，唯独pMC-MT-F1-25的倍数为GAPDH的14.93。
MT is a cysteine-rich, low molecular weight and non-aromatic amino acid protein,andhas a wide range of applications in the field of business and medicine. The traditionalpreparation method of MT has, however restricted its applications,we set out to resolve thepromble with mammary gland bioreactor technology.
First, we use the flanking sequence of the bovine αs1-casein gene,-1660-1437,15628-19476, as regulatory elements of MT. The5' flanking sequence of chickenlysozyme gene and two copies of the chicken β-globin protein insulators were used toeliminate position effects. Through the combination of the three structures, we obtained threeexpression vector pIMC-MT, pIC-MT, pMC-MT.
Secondly, the expression vectors were digested by restriction enzyme and thefragement contain the structure elements were used to generate transgenic mice by pronuclearinjection. F0generation mice were screened by PCR and Southern Blot. The pIMC-MT hadseven positive mice (male mice3,14,16,20, female mice22,24,29),and the pIC-MT hadseven positive mice (male mice2,7,8,10,13, female mice21,27),while pMC-MT had threepositive mice (male mice6,12, female mice20). In order to propagate the number of postivefamale mice, F0postive mice were mated to WT mice. Eight postive female mice wereobtained for PIMC-MT (8,10,11,20,22,23,36,44), three for pIC-MT (6,10,28), and onlyone for pMC-MT (25).
We finally, collected the milk from F0and F1generation, and breast tissue from F1generation. The expression of MT was examined by Western Blot, RT-PCR and Real-timePCR. We could not detect the expression of MT at the protein level. Transcription of MT wasdetected in F1generation of pIMC-MT-F1-11, and all mice of pIC-MT and pMC-MT.Compared to GAPDH, the level of MT transcription was17.87-fold less in pIMC-MT-F1-11,2.25-fold less in pIC-MT-F1-6,4.92-fold less in pIC-MT-F1-10,14.32-fold less inpIC-MT-F1-28. Only in pMC-MT-F1-25,MT was expressed25-fold higher than GAPDH.
引文
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[2] Janssen, A. M., W. van Duijn, et al.(2002)."Prognostic significance of metallothionein in humangastrointestinal cancer." Clin Cancer Res8(6):1889-1896.
[3] Kagi, J. H. and A. Schaffer (1988)."Biochemistry of metallothionein." Biochemistry27(23):8509-8515.
[4] Jin, R., B. H. Bay, et al.(2000)."Metallothionein1E mRNA is highly expressed in oestrogenreceptor-negative human invasive ductal breast cancer." Br J Cancer83(3):319-323.
[5] Haq, F., M. Mahoney, et al.(2003)."Signaling events for metallothionein induction." Mutat Res533(1-2):211-226.
[6] Furey, W. F., A. H. Robbins, et al.(1986)."Crystal structure of Cd,Zn metallothionein." Science231(4739):704-710.
[6] Winge, D. R., K. B. Nielson, et al.(1984)."Structural characterization of the isoforms of neonatal andadult rat liver metallothionein." J Biol Chem259(18):11419-11425.
[7] Winge, D. R., K. B. Nielson, et al.(1984)."Structural characterization of the isoforms of neonatal andadult rat liver metallothionein." J Biol Chem259(18):11419-11425.
[8] Tapiero, H. and K. D. Tew (2003)."Trace elements in human physiology and pathology: zinc andmetallothioneins." Biomed Pharmacother57(9):399-411.
[9] Andrews, G. K.(2000)."Regulation of metallothionein gene expression by oxidative stress and metalions." Biochem Pharmacol59(1):95-104.
[10] Radtke, F., R. Heuchel, et al.(1993)."Cloned transcription factor MTF-1activates the mousemetallothionein I promoter." EMBO J12(4):1355-1362.
[11] Cousins, R. J. and L. M. Lee-Ambrose (1992)."Nuclear zinc uptake and interactions andmetallothionein gene expression are influenced by dietary zinc in rats." J Nutr122(1):56-64.
[12] Cousins, R. J.(1985)."Absorption, transport, and hepatic metabolism of copper and zinc: specialreference to metallothionein and ceruloplasmin." Physiol Rev65(2):238-309.
[13] Squibb, K. S., R. J. Cousins, et al.(1977)."Control of zinc-thionein synthesis in rat liver." Biochem J164(1):223-228.
[14] Richards, M. P. and R. J. Cousins (1975)."Mammalian zinc homeostasis: requirement for RNA andmetallothionein synthesis." Biochem Biophys Res Commun64(4):1215-1223.
[15] Richards, M. P. and R. J. Cousins (1975)."Mammalian zinc homeostasis: requirement for RNA andmetallothionein synthesis." Biochem Biophys Res Commun64(4):1215-1223.
[16] Andrews, G. K.(2000)."Regulation of metallothionein gene expression by oxidative stress and metalions." Biochem Pharmacol59(1):95-104.
[17] Cousins, R. J. and L. M. Lee-Ambrose (1992)."Nuclear zinc uptake and interactions andmetallothionein gene expression are influenced by dietary zinc in rats." J Nutr122(1):56-64.
[18] McKenna, I. M., R. M. Bare, et al.(1996)."Metallothionein gene expression in testicular interstitialcells and liver of rats treated with cadmium." Toxicology107(2):121-130.
[19] Andrews, G. K.(2000)."Regulation of metallothionein gene expression by oxidative stress and metalions." Biochem Pharmacol59(1):95-104.
[20] Cousins, R. J.(1985)."Absorption, transport, and hepatic metabolism of copper and zinc: specialreference to metallothionein and ceruloplasmin." Physiol Rev65(2):238-309.
[21] Kelly, E. J., E. P. Sandgren, et al.(1997)."A pair of adjacent glucocorticoid response elementsregulate expression of two mouse metallothionein genes." Proc Natl Acad Sci U S A94(19):10045-10050.
[22] Samson, S. L. and L. Gedamu (1998)."Molecular analyses of metallothionein gene regulation." ProgNucleic Acid Res Mol Biol59:257-288.
[23] Michalska, A. E. and K. H. Choo (1993)."Targeting and germ-line transmission of a null mutation atthe metallothionein I and II loci in mouse." Proc Natl Acad Sci U S A90(17):8088-8092.
[24] Masters, B. A., E. J. Kelly, et al.(1994)."Targeted disruption of metallothionein I and II genesincreases sensitivity to cadmium." Proc Natl Acad Sci U S A91(2):584-588.
[25] Dunn, M. A. and R. J. Cousins (1989)."Kinetics of zinc metabolism in the rat: effect of dibutyrylcAMP." Am J Physiol256(3Pt1): E420-430.
[26] Thornalley, P. J. and M. Vasak (1985)."Possible role for metallothionein in protection againstradiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxylradicals." Biochim Biophys Acta827(1):36-44.
[27] Cherian, M. G. and M. Nordberg (1983)."Cellular adaptation in metal toxicology andmetallothionein." Toxicology28(1-2):1-15.
[28] Crawford, B. D., M. D. Enger, et al.(1985)."Coordinate amplification of metallothionein I and IIgenes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulatingmetallothionein gene expression." Mol Cell Biol5(2):320-329.
[29] Oh, S. H., J. T. Deagen, et al.(1978)."Biological function of metallothionein. V. Its induction in ratsby various stresses." Am J Physiol234(3): E282-285.
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