CONSTRUCTION AND EXPRESSION IDENTIFICATION OF HUMAN SECRETED APOPTOSIS-RELATED PROTEIN 1 GENE YEAST TWO-HYBRID BAIT VECTOR_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Wei ¹ , HE Guangzhao ² , MA Bing ³

1Department of Medical Cosmetology, the Affiliated Hospital of Xi’an Medical University, Xi’an Shaanxi, 710077, P.R.China;;
2Department of Plastic and Burn Surgery, the First Affiliated Hospital of Chongqing Medical University;;
3Department of Plastic and Burn Surgery, the First Affiliated Hospital of Chengdu Medical College. Corresponding author: MA Bing, E-mail: dmabing@hotmail.com;

Corresponding author：

Keywords：

Secreted apoptosis-related protein 1; Vector construction; Yeast two-hybrid system; Scar

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Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.

Citation： ZHANG Wei,HE Guangzhao,MA Bing. CONSTRUCTION AND EXPRESSION IDENTIFICATION OF HUMAN SECRETED APOPTOSIS-RELATED PROTEIN 1 GENE YEAST TWO-HYBRID BAIT VECTOR. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(12): 1492-1496. doi: Copy

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, 李世荣, 吴军. 分泌型凋亡相关蛋白基因在增生性瘢痕中的分布与作用. 中国美容整形外科杂志, 2008, 19(3): 175-177.
8.	官正华, 刘洋, 杨星华, 等. SARP1在创伤性肌腱瘢痕中分布特点. 重庆医学, 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.	郑高阳, 刘晓颖, 王振英. T载体构建及其构建过程中常见问题的处理方法. 实验室科学, 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. 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, 李世荣, 吴军. 分泌型凋亡相关蛋白基因在增生性瘢痕中的分布与作用. 中国美容整形外科杂志, 2008, 19(3): 175-177.
8. 官正华, 刘洋, 杨星华, 等. SARP1在创伤性肌腱瘢痕中分布特点. 重庆医学, 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. 郑高阳, 刘晓颖, 王振英. T载体构建及其构建过程中常见问题的处理方法. 实验室科学, 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.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND EXPRESSION IDENTIFICATION OF HUMAN SECRETED APOPTOSIS-RELATED PROTEIN 1 GENE YEAST TWO-HYBRID BAIT VECTOR

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