家蝇天蚕素Cec4的原核表达与纯化
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摘要
目的构建家蝇天蚕素(Cecropin)Cec4原核表达质粒,表达及纯化Cec4蛋白。方法从家蝇cDNA文库中筛选家蝇天蚕素基因Cec4,设计并合成特异性引物,PCR扩增Cec4基因,连接克隆载体后转入DH5α感受态细胞,通过重组子鉴定及测序完成目的基因的克隆。用限制性内切酶BamHI和HindⅢ双酶切目的基因和质粒,构建GST表达载体pGEX 4T-1-Cec4,转化到大肠埃希菌BL-21中,利用IPTG诱导表达重组Cec4蛋白,经亲和层析纯化后进行SDS-PAGE电泳分析。结果 PCR扩增获得的Cec4基因全长为192 bp,连接到表达载体pGEX 4T-1获得重组质粒pGEX 4T-1-Cec4,转化大肠埃希菌BL-21后经30℃、0.6 mmol/L的IPTG诱导18 h,表达相对分子质量约为26×10~3上清表达的融合蛋白,亲和层析纯化后的蛋白浓度为0.117 mg/ml。结论成功构建重组质粒pGEX 4T-1-Cec4并表达和纯化获得Cec4蛋白,为其进一步研究奠定了实验基础。
Objective To express and purify the protein Cecropin 4(Cec4) from Musca domestica with a prokaryotic expression vector. Methods Cecropin gene Cec4 was screened from a housefly cDNA library. The Cec4 gene was amplified using PCR with specific primers, ligated into a cloning vector, and then transferred into DH5α competent cells. The target gene was identified using recombinant identification and sequencing. After the target gene and plasmid were double-digested with BamH I and Hind III, the digested Cec4 was ligated with pGEX 4 T-1, and the recombinant plasmid pGEX 4 T-1-Cec4 was transformed into E.coli BL-21. After expression of the plasmid was induced with IPTG, the recombinant Cec4 protein was purified using affinity chromatography and identified using SDS-PAGE. Results A Cec4 fragment 192 bp in length was double-digested and ligated into pGEX 4 T-1, resulting in the recombinant plasmid pGEX 4 T-1-Cec4. E.coli BL-21 harboring the recombinant plasmid was induced to express a protein with 0.6 mmol/L IPTG at 30 ℃, and a 26-ku soluble protein was expressed. The protein concentration after purification with affinity chromatography was 0.117 mg/ml. Conclusion The recombinant plasmid pGEX 4 T-1-Cec4 was successfully constructed and the Cec4 protein was purified. These results have laid the experimental foundation for further research.
Objective To express and purify the protein Cecropin 4(Cec4) from Musca domestica with a prokaryotic expression vector. Methods Cecropin gene Cec4 was screened from a housefly cDNA library. The Cec4 gene was amplified using PCR with specific primers, ligated into a cloning vector, and then transferred into DH5α competent cells. The target gene was identified using recombinant identification and sequencing. After the target gene and plasmid were double-digested with BamH I and Hind III, the digested Cec4 was ligated with pGEX 4 T-1, and the recombinant plasmid pGEX 4 T-1-Cec4 was transformed into E.coli BL-21. After expression of the plasmid was induced with IPTG, the recombinant Cec4 protein was purified using affinity chromatography and identified using SDS-PAGE. Results A Cec4 fragment 192 bp in length was double-digested and ligated into pGEX 4 T-1, resulting in the recombinant plasmid pGEX 4 T-1-Cec4. E.coli BL-21 harboring the recombinant plasmid was induced to express a protein with 0.6 mmol/L IPTG at 30 ℃, and a 26-ku soluble protein was expressed. The protein concentration after purification with affinity chromatography was 0.117 mg/ml. Conclusion The recombinant plasmid pGEX 4 T-1-Cec4 was successfully constructed and the Cec4 protein was purified. These results have laid the experimental foundation for further research.
引文
[1] Liang Y, Wang J, Zhao X, et al. Molecular cloning and characterization of cecropin from the housefly(Musca domestica) and its expression in E. coli[J]. Dev Comp Immunol, 2006, 30(3): 249-57.
[2] Lum KY, Tay ST, Le CF, et al. Activity of novel synthetic peptides against Candida albicans[J]. Sci Rep, 2015, 7(5): 9657.
[3] Leviatan S, Sawada K, Moriyama Y, et al. Combinatorial method for overexpression of membrane proteins in Escherchia coli[J]. J Biol Chen, 2010, 285(31): 23548-56.
[4] Tang T, Huang DW, Zhou CQ, et al. Molecular cloning and expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Musca domestica[J]. Gene, 2012, 4(1): 29.
[5] 龙慧玲, 杨举豪, 彭建, 等. 家蝇天蚕素抗菌肽Cec4对鲍曼不动杆菌抑菌活性的研究[J]. 中华微生物学和免疫学杂志, 2017, 37(12): 891-6.
[6] Ke T, Liang S, Huang J, et al. A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes[J]. BMC Biotechnol, 2012(12): 10.
[7] Yi TH, Sun SY, Huang YB, et al. Prokaryotic expression and mechanism of action of α-helical antimicrobial peptide A20L using fusion tags[J]. BMC Biotechnol, 2015, 15(1): 1-10.
[8] 高安键, 赵若聪, 罗伟芝, 等. 猪蛔虫抗菌肽Cecropin p基因的克隆及其原核表达载体的构建[J]. 江西师范大学学报(自然科学版), 2012, 36(5): 542-6.
[9] 李小波, 王婷婷, 马艳, 等. 家蝇抗菌肽Attacin在毕赤酵母中的异源表达[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(5): 332-6.
[10] 卢敏, 白杰, 魏凤仙, 等. 家蝇抗菌肽diptericin基因在大肠埃希菌中的表达[J]. 生物技术通报, 2016, 32(6): 143-7.
[11] 孙红,薛越, 黄宁. 抗菌肽抗菌机制的研究进展[J]. 中国抗生素杂志, 2013, 38(8): 568-72, 591.
[12] Yang YQ, Wang H, Liang ML,et al. Construction and expression of prokaryotic expression vectors fused with genes of magnaporthe oryzae effector proteins and mCherry[J]. Genet Mol Res, 2015, 14(3): 27-36.
[13] Chameettachal A , Pillai VN, Ali LM,et al. Biochemical and functional characterization of mouse mammary tumor virus Full-Length Pr77Gag expressed in prokaryotic and eukaryotic Cells [J]. Viruses, 2018, 10(6): 334.
[14] 王慧, 齐文静, 何黎, 等. 细粒棘球绦虫成虫相关基因EgM123的克隆、表达及鉴定[J].中国病原生物学杂志, 2017, 12(1): 38-41.
[15] 伍灿, 何树光, 吴翔, 等. 梅毒螺旋体Hsp10蛋白的原核表达与纯化[J]. 中国病原生物学杂志, 2016, 11(12): 1057-61.
[16] Yi Y, Zhao Y, Ma P, et al. Expression and immunogenicity analysis of EsxA protein of Staphylococcus aureus isolated from cow milk[J]. Cn J Biotechnol, 2018, 34(5): 694-702.
[17] Yi TH, Huang YB, Chen YX. Prokaryotic expression and antimicrobial mechanism of XPF-St7-derived α-helical peptides [J]. Pept Sci, 2015, 21(1): 46-52.
[18] Liu Y, Zhan Z, Zhu B, et al. Tandem expression and activity determination of antibacterial peptide Spinosan-C from Paa spinosa[J]. Cn J Biotechnol, 2018, 34(1): 132-9.
[19] 汪以真. 动物源抗菌肽的研究现状和展望[J]. 动物营养学报, 2014, 26(10): 2934-41.
[20] 彭传林, 魏川川, 吴建伟, 等. 家蝇抗真菌肽MAF-1原核表达条件优化及活性验证[J].中国公共卫生, 2015, 31(11): 1420-3.
[21] 王剑, 赵兰华, 李冉辉. 肺炎链球菌Tpx蛋白的原核表达、纯化及活性分析[J]. 中国病原生物学杂志, 2017, 12(6): 485-8, 494.
[2] Lum KY, Tay ST, Le CF, et al. Activity of novel synthetic peptides against Candida albicans[J]. Sci Rep, 2015, 7(5): 9657.
[3] Leviatan S, Sawada K, Moriyama Y, et al. Combinatorial method for overexpression of membrane proteins in Escherchia coli[J]. J Biol Chen, 2010, 285(31): 23548-56.
[4] Tang T, Huang DW, Zhou CQ, et al. Molecular cloning and expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Musca domestica[J]. Gene, 2012, 4(1): 29.
[5] 龙慧玲, 杨举豪, 彭建, 等. 家蝇天蚕素抗菌肽Cec4对鲍曼不动杆菌抑菌活性的研究[J]. 中华微生物学和免疫学杂志, 2017, 37(12): 891-6.
[6] Ke T, Liang S, Huang J, et al. A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes[J]. BMC Biotechnol, 2012(12): 10.
[7] Yi TH, Sun SY, Huang YB, et al. Prokaryotic expression and mechanism of action of α-helical antimicrobial peptide A20L using fusion tags[J]. BMC Biotechnol, 2015, 15(1): 1-10.
[8] 高安键, 赵若聪, 罗伟芝, 等. 猪蛔虫抗菌肽Cecropin p基因的克隆及其原核表达载体的构建[J]. 江西师范大学学报(自然科学版), 2012, 36(5): 542-6.
[9] 李小波, 王婷婷, 马艳, 等. 家蝇抗菌肽Attacin在毕赤酵母中的异源表达[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(5): 332-6.
[10] 卢敏, 白杰, 魏凤仙, 等. 家蝇抗菌肽diptericin基因在大肠埃希菌中的表达[J]. 生物技术通报, 2016, 32(6): 143-7.
[11] 孙红,薛越, 黄宁. 抗菌肽抗菌机制的研究进展[J]. 中国抗生素杂志, 2013, 38(8): 568-72, 591.
[12] Yang YQ, Wang H, Liang ML,et al. Construction and expression of prokaryotic expression vectors fused with genes of magnaporthe oryzae effector proteins and mCherry[J]. Genet Mol Res, 2015, 14(3): 27-36.
[13] Chameettachal A , Pillai VN, Ali LM,et al. Biochemical and functional characterization of mouse mammary tumor virus Full-Length Pr77Gag expressed in prokaryotic and eukaryotic Cells [J]. Viruses, 2018, 10(6): 334.
[14] 王慧, 齐文静, 何黎, 等. 细粒棘球绦虫成虫相关基因EgM123的克隆、表达及鉴定[J].中国病原生物学杂志, 2017, 12(1): 38-41.
[15] 伍灿, 何树光, 吴翔, 等. 梅毒螺旋体Hsp10蛋白的原核表达与纯化[J]. 中国病原生物学杂志, 2016, 11(12): 1057-61.
[16] Yi Y, Zhao Y, Ma P, et al. Expression and immunogenicity analysis of EsxA protein of Staphylococcus aureus isolated from cow milk[J]. Cn J Biotechnol, 2018, 34(5): 694-702.
[17] Yi TH, Huang YB, Chen YX. Prokaryotic expression and antimicrobial mechanism of XPF-St7-derived α-helical peptides [J]. Pept Sci, 2015, 21(1): 46-52.
[18] Liu Y, Zhan Z, Zhu B, et al. Tandem expression and activity determination of antibacterial peptide Spinosan-C from Paa spinosa[J]. Cn J Biotechnol, 2018, 34(1): 132-9.
[19] 汪以真. 动物源抗菌肽的研究现状和展望[J]. 动物营养学报, 2014, 26(10): 2934-41.
[20] 彭传林, 魏川川, 吴建伟, 等. 家蝇抗真菌肽MAF-1原核表达条件优化及活性验证[J].中国公共卫生, 2015, 31(11): 1420-3.
[21] 王剑, 赵兰华, 李冉辉. 肺炎链球菌Tpx蛋白的原核表达、纯化及活性分析[J]. 中国病原生物学杂志, 2017, 12(6): 485-8, 494.