Prokaryotic Expression and Polyclonal Antibody for Ferric Uptake Regulator Prepared from Vibrio vulnificus FJ03-X2
-
摘要: 应用PCR方法克隆了创伤弧菌Vibrio vulnificus FJ03-X2株的铁调基因fur （Ferric uptake regulator），该基因片段大小为450 bp，编码149个氨基酸；以pET32a为表达载体，构建了原核表达质粒pET32a-FUR，表达质粒测序结果表明目的基因与GenBank中报道的创伤弧菌fur基因的同源性达98%以上；诱导表达获得可溶性的重组表达蛋白rFUR。镍离子金属螯合亲和层析介质（Ni-NTA）纯化rFUR，SDS-PAGE电泳分析其分子量约33 kD。以纯化后的融合蛋白rFUR为抗原，4次免疫SD大鼠，制备抗rFUR蛋白大鼠多克隆抗体。用ELISA方法检测鼠多克隆抗体的效价达到1：256 000，表明融合蛋白rFUR具有良好的免疫原性。Abstract: Ferric uptake regulator (fur) gene from Vibrio vulnificus FJ03-X2 was cloned, and inserted into the prokaryotic expression vector, pET32a. Coding sequence of the gene contained 450 bps, encoding 149 amino acids. Its sequencing showed a greater than 98% homology with that of the fur gene from GenBank. Subsequently, the plasmid, pET32a-FUR, was transformed into E. coli BL21 to express the recombinant protein, rFUR. The soluble rFUR was then subject to the Ni-NTA His Binding affinity purification. The purified rFUR was injected into SD rats to produce polyclonal antibody. The obtained highly specific antibodies of FUR was confirmed by ELISA assay.
-
Key words:
- Vibrio vulnificus /
- ferric uptake regulator /
- cloning /
- prokaryotic expression /
- polyclonal antibody
-
图 1 基因的PCR扩增电泳结果
1~3 为fur基因；4为阴性对照；5为DL2000分子量标准。
Figure 1. PCR amplification of fur gene
图 2 质粒的双酶切鉴定
1为 λ-EcoT14 分子量标准； 2~6为重组子酶切结果；7为 DL2000分子量标准。
Figure 2. Results of dual-enzyme digestion
图 4 Ni-NTA纯化的rFUR蛋白的SDS-PAGE结果
注：1为 BL21/pET32a空载诱导全菌蛋白；2为BL21/pET32a-FUR未诱导的全菌蛋白；3为BL21/pET32a-FUR经诱导后超声的菌体破碎物上清蛋白；4为纯化后的rFUR融合蛋白；5为蛋白质低分子量标准。
Figure 4. SDS-PAGE analysis of purified rFUR protein by Ni-NTA
表 1 ELISA 测定抗体效价(λ=490nm)
Table 1. Determination of antibody titer by ELISA
项目 1∶4000 1∶8000 1∶16000 1∶32000 1∶64000 1∶128000 1∶256000 抗血清 2.5755 2.3855 2.2835 2.034 1.722 1.176 0.783 阴性血清 0.1335 0.116 0.1135 0.1295 0.1095 0.116 0.1155 
下载: 导出CSV
-
[1] GUERRERO A,GOMEZ G R B,WONG-CHANG I,et al. Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico[J]. Int J Environ Health Res. 2015, 25(6):614-627. doi: 10.1080/09603123.2014.1003038 [2] DONASCIMENTO S, DOS FERNANDES V R,THEOPHILO G N,et al. Vibrio vulnificus as a health hazard for shrimp consumers[J]. Rev Inst Med Trop Sao Paulo, 2001, 43(5):263-266. doi: 10.1590/S0036-46652001000500005 [3] 许斌福,林天龙,董传甫. 鳗鲡创伤弧菌的分子鉴定[J].中国人兽共患病杂志,2005, 11(21):995-997. http://www.cnki.com.cn/Article/CJFDTOTAL-ZRSZ200511018.htm [4] 辜良斌,徐力文,王雨,等. 从珍珠龙趸体内分离的1株创伤弧菌的鉴定及耐药性分析[J]. 大连海洋大学学报, 2016, 31(2):162-167. http://www.cnki.com.cn/Article/CJFDTOTAL-DLSC201602008.htm [5] 于兰萍,王斌,李艳,等.大菱鲆出血性败血症病原菌的分离与鉴定[J]. 大连水产学院学报, 2008, 23(5):335-339. http://www.cnki.com.cn/Article/CJFDTOTAL-DLSC200805002.htm [6] LOHITH G K, KINGSTON J J, SINGH A K, et al. Evaluation of recombinant leukocidin domain of VvhA exotoxin of Vibrio vulnificus as an effective toxoid in mouse model[J]. Immunol Lett. 2015, 167(1):47-53. doi: 10.1016/j.imlet.2015.06.015 [7] LEE S J, JUNG Y H, OH S Y, et al. Vibrio vulnificus VvhA induces NF-kappaB-dependent mitochondrial cell death via lipid raft-mediated ROS production in intestinal epithelial cells[J]. Cell Death Dis, 2015, (6):1655. http://cn.bing.com/academic/profile?id=2012495668&encoded=0&v=paper_preview&mkt=zh-cn [8] KIM B S, SATCHELL K J. MARTX effector cross kingdom activation by Golgi-associated ADP-ribosylation factors[J]. Cell Microbiol, 2016,18(8):1078-1093. doi: 10.1111/cmi.v18.8 [9] ZIOLO K J, JEONG H G, KWAK J S, et al. Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice[J]. Infect Immun, 2014, 82(5):2148-2157. doi: 10.1128/IAI.00017-14 [10] LEE S J, JUNG Y H, OH S Y, et al. Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells[J]. Cell Death Di, 2015, (6):e1787. http://cn.bing.com/academic/profile?id=913659548&encoded=0&v=paper_preview&mkt=zh-cn [11] LEE K J, KIM J A, HWANG W, et al. Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus[J]. Mol Microbiol, 2013, 90(4):841-857. doi: 10.1111/mmi.12401 [12] LEE H J, KIM J A, LEE M A, et al. Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus:repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases[J]. Mol Microbiol, 2013, 88(4):813-826. doi: 10.1111/mmi.2013.88.issue-4 [13] SONGLIN G, PANPAN L, JIANJUN F, et al. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata)[J]. Fish Shellfish Immunol, 2015, 43(2):477-484. doi: 10.1016/j.fsi.2015.01.017 [14] KIM I H,WEN Y,SON J S, et al. The fur-iron complex modulates expression of the quorum-sensing master regulator, SmcR, to control expression of virulence factors in Vibrio vulnificus[J]. Infect Immun, 2013, 81(8):2888-2898. doi: 10.1128/IAI.00375-13 [15] LEE H J,KIM J A,LEE M A, et al. Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus:repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases[J]. Mol Microbiol, 2013, 88(4):813-826. doi: 10.1111/mmi.2013.88.issue-4 [16] MACHADOH,GRAM L. The fur gene as a new phylogenetic marker for Vibrionaceae species identification[J]. Appl Environ Microbiol, 2015, 81(8):2745-2752. doi: 10.1128/AEM.00058-15 [17] 田丁,许斌福,林能峰. 创伤弧菌外膜蛋白免疫刺激复合物对欧洲鳗鲡的免疫保护性分析[J]. 水生生物学报, 2010, 34(2):431-435. http://www.cnki.com.cn/Article/CJFDTOTAL-SSWX201002029.htm -
点击查看大图
图(4) / 表(1)
计量
- 文章访问数: 1420
- HTML全文浏览量: 162
- PDF下载量: 250
- 被引次数: 0
