Construction of Prokaryotic Expression Plasmid pET32a-AcGFPl of Green Fluorescent Protein and Their High Efficient Expression in Escherichia Coli
HUO Hai-long1,2, ZHAO Yue1, WANG Rui1, Hou Hui-fang3, LI Wei-zhen4, ZHANG Yong-yun5, LIU Li-xian1, WANG Pei4, HUO Jin-long4
1. Department of Husbandry and Veterinary, Yunnan Vocational and Technical College of Agriculture, Kunming 650031, China;
2. Faculty of Life Science, Yunnan University, Kunming 650091, China;
3. Bureau of Agricultural Machinery Administration of Xinzhou City, Xinzhou 034000, China;
4. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China;
5. Teaching Demonstration Center of the Basic Experiments of Agricultural Majors, Yunnan Agricultural University, Kunming 650201, China
为了构建绿色荧光蛋白AcGFP1基因的原核表达载体pET32a-AcGFP1，并诱导使其在大肠杆菌中高效表达，本研究以pIRES-AcGFP1质粒为模板，采用PCR技术特异性扩增绿色荧光蛋白AcGFP1基因，通过酶切和连接使其与原核表达载体pET32a(+)构成重组子，经PCR、酶切和测序鉴定后，重组质粒转化大肠杆菌Rosetta(DE3)，用不同浓度的异丙基硫代半乳糖苷(IPTG)诱导表达绿色荧光蛋白，经15%SDS-PAGE鉴定，结果显示：重组质粒pET32a-AcGFP1中的AcGFP1序列与Clontech公司的pIRES-AcGFP1质粒的AcGFP1序列完全一致，表明本实验已成功构建了含有绿色荧光蛋白基因AcGFP1的原核表达质粒。此外，pET32a-AcGFP1转化Rosetta(DE3)，经不同浓度的IPTG诱导，SDS-PAGE检测均获得高效表达，为今后构建pET32a(+)-TAT-AcGFP1融合表达载体以及深入研究TAT的细胞膜穿透作用的机制奠定基础。 To construct prokaryotic expression vector pET32a-AcGFP1 and make it express efficiently in E. coli cell, the AcGFP1 gene of green fluorescent protein was amplified from pIRES-AcGFP1 plasmid with PCR method, and was inserted into prokaryotic expression vector pET32a (+) by restriction enzyme digestion. After identified by PCR, restriction enzyme digestion and sequencing, the AcGFP1 gene was induced with different concentrations' isopropyl-(3-D-thiogalactopyranoside (IPTG) and detected on 15% SDS-PAGE. Results showed that the sequence of AcGFP1 gene in pET32a-AcGFP1 recombinant plasmid is consistent with that AcGFP1 sequence in pIRES-AcGFP1 plasmid from Clontech Ltd, proving that the prokaryotic expression vector with AcGFP1 gene of green fluorescent protein is constructed successfully. Moreover, all of the pET32a-AcGFP1 plasmids transformed in E. coli Rosetta (DE3) were high efficient expression after inducing by different concentrations of IPTG. Results above will provide a basis on constructing pET32a(+)-TAT-AcGFP1 fusion expression vector and studying deeply mechanism of TAT cell membrane penetration.
HUO Hai-long, ZHAO Yue, WANG Rui, Hou Hui-fang, LI Wei-zhen, ZHANG Yong-yun, LIU Li-xian, WANG Pei, HUO Jin-long .Construction of Prokaryotic Expression Plasmid pET32a-AcGFPl of Green Fluorescent Protein and Their High Efficient Expression in Escherichia Coli[J] Journal of Yunnan Agricultural University, 2012,V27(6): 820-825
 PRASHER D C. Primary structure of Aequorea victoria green-fluorescent protein [J]. Gene,1992,111：229-233. CHALFIE M,TU Y,EUSKIRCHEN G,et a1. Green fluorescent proteins as a marker for gene expression [J]. Science,1994,263(1)：802-805. CUBITT A B,HEIM R,ADAMS S R. Understanding, improving and using green fluorescent proteins [J]. Trends in Biochemical Sciences,1995,20：448-455. BOONYARATTANAKALIN S, ATHAVANKAR S, SUN Q,et al. Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell penetrating peptides [J]. Journal of -387. miKONEN R,MATILAINEN H,RAJ ALA N,et al. Functional display of an alpha integrin-specific motif (RKK) on the surface of baculovirus particles [J]. Technology in Cancer Research & Treatment,2005,4(4)：437-445. EGUCHI A,AKUTA T,OKUYAMA H,et a1. Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells [J]. Journal of Biological Chemistry,2001,276(28)：26204-26210. BECKER-HAPAK M,MCALLISTER S S,DOWDY S F. TAT-mediated protein transduction into mammalian cells .Methods,2001,24(3)：247-256. CARON N J,TORRENTE Y,CAMIRAND G,et al. In- tracellular delivery of a Tat-eGFP fusion protein into muscle cells [J]. Molecular Therapy,2001,3(3)：310-318. LIU AX,ZHANG S B,REN DT,et al. Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)[J]. Cell Research,2004,14(5)：407-414. KOHLER R H,CAO J,ZIPFEL W R,et al. Exchange of Protein Molecules Through Connections Between Higher Plant Plastids [J]. Science,1997,276(5321)：2039-2042. CHIU W,NOWA Y,ZENG W,et al. Engineered GFP as a vital reporter in plants [J]. Current Biology,1996,6(3)：325-330. VALDIVIAA R H,HROMOCKYJA A E,MONACKA D, et al. Applications for green fluorescent protein (GFP) in the study of host pathogen interaction [J]. Gene,173(1)：47-52.