AccRoyalisin重组表达及产物抑菌活性研究
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摘要
蜂王浆(royal jelly)是蜜蜂工蜂咽下腺、上颚腺和脑后腺等腺体(俗称王浆腺)分泌蜂王浆的乳状物质,是1-3龄工蜂幼虫及蜂王的终生食料,它含有丰富的生物活性成分,多种生理活性和营养保健功能。中华蜜蜂(Apis cerana cerana)是原产于我国的土著具蜂种,具有明显优于西方蜜蜂(Apis mellifera)的抗病性。近期研究表明,中华蜜蜂抗病性优于西方蜜蜂以及蜂王浆不易变质的特性都与抗菌肽royalisin有关。
此前,本实验室已完成了对中华蜜蜂抗菌肽Acc-Royalisin的原核表达及表达产物抑菌活性初步研究。本研究重点进行了原核表达放大试验,产物规模化分离纯化。在此基础上,进行了原核表达产物抑菌谱、代表菌最低抑菌浓度、抑菌稳定性、抑菌机理的系统研究。
同时,进行了Acc-Royalisin基因真核表达载体构建。首先,以含Acc-Royalisin基因的载体为模板,采用引入编码6×His+EK DNA序列的5-P引物,和编码Acc-Royalisin基因的3-P引物,进行目的基因PCR扩增。然后将引入6×His+EK DNA序列的目的基因连结到pGEX-4T-2载体的GST基因3'端。最后,以此插入GST-6×His-EK-AccRoyalisin序列的载体pGEX-4T-2-AccRoyalisin为模板,采用根据此重组序列和酵母表达载体pPIC9K的相应酶切位点Snab I和NotⅠ设计的5-P和3-P引物,进行目的基因PCR扩增,得到长度约850 bp的DNA片段。将纯化的PCR产物和酵母表达载体pPIC9K分别用Snab I和Not I双酶切,酶切产物再分别作电泳检测和割胶回收,连接,转化DH5α感受态细胞,培养筛选出重组AccRoyalisin-pPIC9K阳性克隆。将AccRoyalisin-pPIC9K线性化,电击转化毕彻酵母GS115。抽提酵母基因组DNA,进行PCR鉴定,筛选出含目的基因的重组子。
对重组酵母行诱导表达,表达产物SDS-PAGE电泳分析。结果表明,表达产物在30 kDa处具有特异性条带。以GST多克隆抗体为一抗进一步作Western blot检测,结果检测到目的蛋白印迹,说明AccRoyalisin基因在酵母菌中也已表达成功。所表达的多肽经纯化获得单一蛋白。抑菌活性检测结果表明,重组酵母表达的AccRoyalisin对革兰氏阳性细菌枯草芽孢杆菌和黄色微球菌具有抑制活性。
Royal jelly (RJ) is a glandular secretion produced by honeybees nurse to nourish honeybee larvae during their first three days of life and as the sole food for the queen during its life span. It contains rich biological active composition, and with much nutritional and physical functions. The Chinese honeybee (Apis cerana cerana) is a native honeybee in China, and its disease resistance is superior to the Western honeybee (Apis mellifera). Recent researches have shown that the disease resistance and the characteristics of non-perishable royal jelly are related to the peptide royalisin.
Our laboratory has previously completed the expression of the Chinese honeybee peptide, AccRoyalisin and the preliminary studies on the antibacterial activity of its recombinant expression products in E. coli. This study focused on the enlarge fermentation tests of the prokaryotic expression, the large-scale expression of the products, and the separation and purification of the products. Based on these reserch, the systematic study of establishment of prokaryotic expression products on the inhibitory spectrum, minimum inhibitory concentration test (MIC) for representative strains, antibacterial stability experiments and antibacterial mechanism experiments were included.
Firstly, adopting primers 5-P contained 6×His-EK DNA and 3-P, and AccRoyalisin-pGEM vector as the template, the recombinant AccRoyalisin was amplified with PCR reaction. Secondly, the PCR product was linked with GST whithin pGEX-4T-2 vector. Finally, using the recombinant vector, pGEX-4T-2-AccRoyalisin as template, the DNA fragment GST-6×His-EK-AccRoyalisin with 850 bp in length was amplified and inserted into yeast expression vector pPIC9K. The linilized AccRoyalisin-pPIC9K was transformef into of Pichia yeast GS115 with electroporation. Through PCR identification with the DNA genome extrcted from the yeast clones, the recombinant yeast with purpose gene was identified.
The recombinant yeast clones were induced to express peptides. SDS-PAGE analysis showed that the expression product possessed a specific band wih 30 kDa in molecular weight. Western blot analysis using the GST polyclonal antibody as the first antibody proved that GST-AccRoyalisin was expressed in yeast successfuly. A a single peptide band was purified from the expressed product.
Antibacterial detection showed that the recombinant AccRoyalisin expressed in yeast possessed inhibitory activity against Gram-positive bacterias, such as Bacillus subtilis and Micrococcus lysodeikticus.
此前,本实验室已完成了对中华蜜蜂抗菌肽Acc-Royalisin的原核表达及表达产物抑菌活性初步研究。本研究重点进行了原核表达放大试验,产物规模化分离纯化。在此基础上,进行了原核表达产物抑菌谱、代表菌最低抑菌浓度、抑菌稳定性、抑菌机理的系统研究。
同时,进行了Acc-Royalisin基因真核表达载体构建。首先,以含Acc-Royalisin基因的载体为模板,采用引入编码6×His+EK DNA序列的5-P引物,和编码Acc-Royalisin基因的3-P引物,进行目的基因PCR扩增。然后将引入6×His+EK DNA序列的目的基因连结到pGEX-4T-2载体的GST基因3'端。最后,以此插入GST-6×His-EK-AccRoyalisin序列的载体pGEX-4T-2-AccRoyalisin为模板,采用根据此重组序列和酵母表达载体pPIC9K的相应酶切位点Snab I和NotⅠ设计的5-P和3-P引物,进行目的基因PCR扩增,得到长度约850 bp的DNA片段。将纯化的PCR产物和酵母表达载体pPIC9K分别用Snab I和Not I双酶切,酶切产物再分别作电泳检测和割胶回收,连接,转化DH5α感受态细胞,培养筛选出重组AccRoyalisin-pPIC9K阳性克隆。将AccRoyalisin-pPIC9K线性化,电击转化毕彻酵母GS115。抽提酵母基因组DNA,进行PCR鉴定,筛选出含目的基因的重组子。
对重组酵母行诱导表达,表达产物SDS-PAGE电泳分析。结果表明,表达产物在30 kDa处具有特异性条带。以GST多克隆抗体为一抗进一步作Western blot检测,结果检测到目的蛋白印迹,说明AccRoyalisin基因在酵母菌中也已表达成功。所表达的多肽经纯化获得单一蛋白。抑菌活性检测结果表明,重组酵母表达的AccRoyalisin对革兰氏阳性细菌枯草芽孢杆菌和黄色微球菌具有抑制活性。
Royal jelly (RJ) is a glandular secretion produced by honeybees nurse to nourish honeybee larvae during their first three days of life and as the sole food for the queen during its life span. It contains rich biological active composition, and with much nutritional and physical functions. The Chinese honeybee (Apis cerana cerana) is a native honeybee in China, and its disease resistance is superior to the Western honeybee (Apis mellifera). Recent researches have shown that the disease resistance and the characteristics of non-perishable royal jelly are related to the peptide royalisin.
Our laboratory has previously completed the expression of the Chinese honeybee peptide, AccRoyalisin and the preliminary studies on the antibacterial activity of its recombinant expression products in E. coli. This study focused on the enlarge fermentation tests of the prokaryotic expression, the large-scale expression of the products, and the separation and purification of the products. Based on these reserch, the systematic study of establishment of prokaryotic expression products on the inhibitory spectrum, minimum inhibitory concentration test (MIC) for representative strains, antibacterial stability experiments and antibacterial mechanism experiments were included.
Firstly, adopting primers 5-P contained 6×His-EK DNA and 3-P, and AccRoyalisin-pGEM vector as the template, the recombinant AccRoyalisin was amplified with PCR reaction. Secondly, the PCR product was linked with GST whithin pGEX-4T-2 vector. Finally, using the recombinant vector, pGEX-4T-2-AccRoyalisin as template, the DNA fragment GST-6×His-EK-AccRoyalisin with 850 bp in length was amplified and inserted into yeast expression vector pPIC9K. The linilized AccRoyalisin-pPIC9K was transformef into of Pichia yeast GS115 with electroporation. Through PCR identification with the DNA genome extrcted from the yeast clones, the recombinant yeast with purpose gene was identified.
The recombinant yeast clones were induced to express peptides. SDS-PAGE analysis showed that the expression product possessed a specific band wih 30 kDa in molecular weight. Western blot analysis using the GST polyclonal antibody as the first antibody proved that GST-AccRoyalisin was expressed in yeast successfuly. A a single peptide band was purified from the expressed product.
Antibacterial detection showed that the recombinant AccRoyalisin expressed in yeast possessed inhibitory activity against Gram-positive bacterias, such as Bacillus subtilis and Micrococcus lysodeikticus.
引文
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Brogden K A, De L A, Bland J, et al. Isolation of an oviae pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica [J]. Proc Natl Acad Sci USA 1996,93(1):412-416.
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Bilikova K, Wu G, Simuth J. Isolation of a peptide fraction from honeybee royal jelly as a potential antifoulbrood factor [J]. Apidologie,2001,32 (3):275-283.
Birkemo G A, Luders T, Andersen O, et al. Nissen-Meyer J. Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut(Hippoglossus hippoglossus L.) [J]. Biochimica et Biophysica Acta,2003,1646:207-215.
Boman H G, Nilsson, Rasmuson B. Inducible antibacterial defence system in Drosophila [J]. Nature,1972,237:232-235
Boutigue M H, Lugardon K, Goumon Y, et al. Antibacterial and antifungal peptides derived from chromogranins and proenkephalin-A:from structure to biological aspects[J]. Adv Exp Med Biol,2000,482:299-315.
Brogden K A, Ackermann M, Huttner K M. Detection of anionic antimicrobial peprides in ovine bronchoalveolar lavage fluid and respiratory epithelium [J]. Infect Immun.1998,66(12):5948-5954.
Brogden K A, Ackermann M R, McCray P B, et al. Differences in the concentrations of small, anionic, antimicrobial peptides in bronchoalveolar lavage fluid and in respiratory epithelia of patients with and without cystic fibrosis [J]. Infect Immun. 1999,67(8):4256-4259.
Brogden K A, De L A, Bland J, et al. Isolation of an oviae pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica [J]. Proc Natl Acad Sci USA 1996,93(1):412-416.
Bulet P, Dimareq J L, Hetru C, et al. A novel inducible antibacterial peptide of Drosophila carries an o-glycosylated substitution [J]. Biol Chem 1993,268(20): 14893-14897.
Carl L, Philippe B, Maurice, et al. Insect immunity:Isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti [J]. Insect Biochemistry and Molecular Biology 1995,25(7):867-873
Casteels P, Ampe C, Jacobs F, et al. Apidaocins:antibacterial peptides from honeybees [J]. EMBO.1989.8(8):2387-2391.
Casteels P, Ampe C, Riviere L, et al. Isolation and characterization of abaecin, a major a antibacterial response peptide in the honeybees (Apis mellifera) [J]. Eur Biochem,1990.187 (2):381-386.
Carlsson A, Bennich H. Attacin, an antibacterial protein from Hyalophora cecropia, inhibit syntheseis of outer membrane proteins in Echerichia coli by interfering with omp gene teanscription [J]. Infect and Immunity,1991,59(9):3040-3045.
Casteels P, Romagnolo J, Castle M, et al. Erdjument Bromage H, Tempst P. Biodiversity of apidaecin-type peptide antibiotics. Prospects of manipulating the antibacterial spectrum and combating acquired resistance [J]. Biol Chem.1994, 269(42):26107-26115.
Chen P, Novak J, Kirk M, et al. Structure-activity study of the lantibiotic mutacin Ⅱ from Streptococcus mutans T8 by a gene replacement strategy [J]. Appl Environ Microbiol.1998,64(7):2335-2340.
Cereghino J L; Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiol Rev.2000,24,45-66.
Cociancich S, Bulet P, Hetru C, et al. The induced antibacterial peptides of insects [J]. Parasitol Today 1994,10(4):132.
Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris [J]. Bio-Technology,1993,11:905-910.
Easton D M, Nijnik A, Mayer M L, et al. Potential of immunomodulatory host defense peptides as novel antiinfectives [J]. Trends Biotechnol,2009,27(10): 582-590.
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