張偉 1 , 賀光照 2 , 馬兵 3
  • 1西安醫(yī)學(xué)院附屬醫(yī)院醫(yī)學(xué)美容科（西安，710077）;;
  • 2 重慶醫(yī)科大學(xué)附屬第一醫(yī)院整形美容燒傷科;;
  • 3 成都醫(yī)學(xué)院附屬第一醫(yī)院燒傷整形科;

目的 構(gòu)建人分泌的凋亡相關(guān)蛋白1（secreted apoptosis-related protein 1，SARP1）基因酵母雙雜交誘餌載體，為鑒定SARP1基因相互作用蛋白、探討SARP1基因在瘢痕組織中的生物學(xué)功能奠定基礎(chǔ)。 方法根據(jù)GenBank中SARP1的mRNA序列，設(shè)計(jì)分別帶有NdeⅠ和SalⅠ酶切位點(diǎn)的上、下游引物，人工合成SARP1基因片段，擴(kuò)增SARP1基因片段，構(gòu)建含pGBKT7-SARP1基因的重組質(zhì)粒，用NdeⅠ和SalⅠ進(jìn)行雙酶切、PCR，凝膠電泳及測(cè)序。用醋酸鋰法將序列正確的重組質(zhì)粒pGBKT7-SARP1（卡那霉素抗性篩選）轉(zhuǎn)化入AH109酵母菌株，在缺陷型培養(yǎng)基上觀察pGBKT7-SARP1在AH109中的表達(dá)情況。 結(jié)果 SARP1基因擴(kuò)增后的片段大小符合預(yù)期，并成功克隆入pGBKT7載體中；酶切后凝膠電泳及DNA測(cè)序顯示pGBKT7-SARP1基因重組載體構(gòu)建正確，對(duì)酵母菌株AH109無毒性，且不具自主激活報(bào)告基因的效應(yīng)。 結(jié)論成功構(gòu)建了pGBKT7-SARP1基因酵母雙雜交誘餌載體。

引用本文： 張偉,賀光照,馬兵. 人分泌的凋亡相關(guān)蛋白1基因酵母雙雜交誘餌載體的構(gòu)建及表達(dá)鑒定. 中國修復(fù)重建外科雜志, 2012, 26(12): 1492-1496. doi: 復(fù)制

1. Melkonyan HS, Chang WC, Shapiro JP, et al. SARPs: a family of secreted apoptosis-related proteins. Proc Natl Acad Sci U S A, 1997, 94(25): 13636-13641.
2. Wu J, Ma B, Yi S, et al. Gene expression of early hypertrophic scar tissue screened by means of cDNA microarrays. J Trauma, 2004, 57(6): 1276-1286.
3. Chen W, Fu X, Sun X, et al. Analysis of differentially expressed genes in keloids and normal skin with cDNA microarray. J Surg Res, 2003, 113(2): 208-216.
4. Nojima M, Suzuki H, Toyota M, et al. Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer. Oncogene, 2007, 26(32): 4699-4713．.
5. Grotendorst GR, Duncan MR. Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J, 2005, 19(7): 729-738.
6. Desmoulière A, Darby IA, Gabbiani G. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest, 2003, 83(12): 1689-1707.
7. 王珍祥, 李世榮, 吳軍. 分泌型凋亡相關(guān)蛋白基因在增生性瘢痕中的分布與作用. 中國美容整形外科雜志, 2008, 19(3): 175-177.
8. 官正華, 劉洋, 楊星華, 等. SARP1在創(chuàng)傷性肌腱瘢痕中分布特點(diǎn). 重慶醫(yī)學(xué), 2009, 38(13): 1626-1227.
9. Linge C, Richardson J, Vigor C, et al. Hypertrophic scar cells fail to undergo a form of apoptosis specific to contractile collagen-the role of tissue transglutaminase. J Invest Dermatol, 2005, 125(1): 72-82.
10. Wang DR, Tang D. Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening. World J Gastroenterol, 2008, 14(4): 524-531.
11. Oberwalder M, Wonter C, Wöntner C, et al. SFRP2 methylation in fecal DNA—a marker for colorectal polyps. Int J Colorectal Dis, 2008, 23(1): 15-19.
12. Tang D, Wang DR, Li HB. Combination analysis of hypermethylated SFRP1 and SFRP2 gene in fecal as a novel epigenetic biomarker panel for colorectal cancer screening. Journal of Nanjing Medical University, 2008, 22(2): 96-101.
13. Ellies DL, Church V, Francis-West P, et al. The Wnt antagonist cSFRP2 modulates programmed cell death in the developing hindbrain. Development, 2000, 127(24): 5285-5295.
14. Ye X, Lu H, Huo K, et al. Finding a novel interacting protein of the hepatic carcinoma related gene MIP: NF-κB essential modulator (NEMO). Oncol Rep, 2011, 25(1): 231-235．.
15. Kodama Y, Suetsugu N, Kong SG, et al. Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis. Proc Natl Acad Sci U S A, 2010, 107(45): 19591-19596．.
16. Parent A, Roy SJ, Iorio-Morin C, et al. ANKRD13C acts as a molecular chaperone for G protein-coupled receptors. J Biol Chem, 2010, 285(52): 40838-40851.
17. Tripathi LP, Kataoka C, Taguwa S, et al. Network based analysis of hepatitis C virus Core and NS4B protein interactions. Mol Biosyst, 2010, 6(12): 2539-2553．.
18. 鄭高陽, 劉曉穎, 王振英. T載體構(gòu)建及其構(gòu)建過程中常見問題的處理方法. 實(shí)驗(yàn)室科學(xué), 2009, 2(1): 116-117.
19. Zhou MY, Clark SE, Gomez-Sauchez CE. Universal cloning method by TA strategy. Biotechniques, 1995, 19(1): 34-35.
20. Bartkowiak B, Liu P, Phatnani HP, et al. CDK12 is a tran-scription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctkl. Genes Dev, 2010, 24(20): 2303-2316.
  1. 1. Melkonyan HS, Chang WC, Shapiro JP, et al. SARPs: a family of secreted apoptosis-related proteins. Proc Natl Acad Sci U S A, 1997, 94(25): 13636-13641.
  2. 2. Wu J, Ma B, Yi S, et al. Gene expression of early hypertrophic scar tissue screened by means of cDNA microarrays. J Trauma, 2004, 57(6): 1276-1286.
  3. 3. Chen W, Fu X, Sun X, et al. Analysis of differentially expressed genes in keloids and normal skin with cDNA microarray. J Surg Res, 2003, 113(2): 208-216.
  4. 4. Nojima M, Suzuki H, Toyota M, et al. Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer. Oncogene, 2007, 26(32): 4699-4713．.
  5. 5. Grotendorst GR, Duncan MR. Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J, 2005, 19(7): 729-738.
  6. 6. Desmoulière A, Darby IA, Gabbiani G. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest, 2003, 83(12): 1689-1707.
  7. 7. 王珍祥, 李世榮, 吳軍. 分泌型凋亡相關(guān)蛋白基因在增生性瘢痕中的分布與作用. 中國美容整形外科雜志, 2008, 19(3): 175-177.
  8. 8. 官正華, 劉洋, 楊星華, 等. SARP1在創(chuàng)傷性肌腱瘢痕中分布特點(diǎn). 重慶醫(yī)學(xué), 2009, 38(13): 1626-1227.
  9. 9. Linge C, Richardson J, Vigor C, et al. Hypertrophic scar cells fail to undergo a form of apoptosis specific to contractile collagen-the role of tissue transglutaminase. J Invest Dermatol, 2005, 125(1): 72-82.
  10. 10. Wang DR, Tang D. Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening. World J Gastroenterol, 2008, 14(4): 524-531.
  11. 11. Oberwalder M, Wonter C, Wöntner C, et al. SFRP2 methylation in fecal DNA—a marker for colorectal polyps. Int J Colorectal Dis, 2008, 23(1): 15-19.
  12. 12. Tang D, Wang DR, Li HB. Combination analysis of hypermethylated SFRP1 and SFRP2 gene in fecal as a novel epigenetic biomarker panel for colorectal cancer screening. Journal of Nanjing Medical University, 2008, 22(2): 96-101.
  13. 13. Ellies DL, Church V, Francis-West P, et al. The Wnt antagonist cSFRP2 modulates programmed cell death in the developing hindbrain. Development, 2000, 127(24): 5285-5295.
  14. 14. Ye X, Lu H, Huo K, et al. Finding a novel interacting protein of the hepatic carcinoma related gene MIP: NF-κB essential modulator (NEMO). Oncol Rep, 2011, 25(1): 231-235．.
  15. 15. Kodama Y, Suetsugu N, Kong SG, et al. Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis. Proc Natl Acad Sci U S A, 2010, 107(45): 19591-19596．.
  16. 16. Parent A, Roy SJ, Iorio-Morin C, et al. ANKRD13C acts as a molecular chaperone for G protein-coupled receptors. J Biol Chem, 2010, 285(52): 40838-40851.
  17. 17. Tripathi LP, Kataoka C, Taguwa S, et al. Network based analysis of hepatitis C virus Core and NS4B protein interactions. Mol Biosyst, 2010, 6(12): 2539-2553．.
  18. 18. 鄭高陽, 劉曉穎, 王振英. T載體構(gòu)建及其構(gòu)建過程中常見問題的處理方法. 實(shí)驗(yàn)室科學(xué), 2009, 2(1): 116-117.
  19. 19. Zhou MY, Clark SE, Gomez-Sauchez CE. Universal cloning method by TA strategy. Biotechniques, 1995, 19(1): 34-35.
  20. 20. Bartkowiak B, Liu P, Phatnani HP, et al. CDK12 is a tran-scription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctkl. Genes Dev, 2010, 24(20): 2303-2316.