乳酸片球菌素pedA基因的克隆与表达
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摘要
细菌素作为一种天然生物防腐剂，用于食品防腐以提高食品安全性和延长食品货架期。为解决天然菌株细菌素产量较低的问题，本课题采用基因工程技术，克隆乳酸片球菌的片球菌素pedA基因，建立大肠杆菌片球菌素基因pedA表达系统，以期获得片球菌素的高效表达。
以乳酸片球菌基因组为模板，采用PCR法，扩增出了片球菌素的结构基因pedA。将扩增的pedA基因连接到pMD18-T载体，转化至大肠杆菌DH5α。提取含有pedA基因的pMD18-T重组质粒，Bgl II和Xhol I双酶切获得pedA基因，与经同样双酶切后的表达质粒pET32a(+)连接，先后转化至大肠杆菌DH5α和大肠杆菌BL21(DE3)，用PCR法筛选阳性克隆菌株后，选取其中的一株阳性克隆菌株，提取重组质粒，进行pedA基因测序，发现该重组质粒含有pedA基因，其核苷酸序列与NCBI公布的片球菌素pedA基因（GenBank AY083244）同源性为100%。该菌株在含有1mmol/L IPTG的培养基中，37℃培养4小时后，高效表达了预期大小的22kDa硫氧还蛋白和片球菌素的融合蛋白。
重组菌培养物经超声破碎后离心，分别取上清和沉淀物用SDS-PAGE进行分析，由于在沉淀物SDS-PAGE电泳结果中有预期大小的融合蛋白条带，表明表达的蛋白为包涵体。为获得有活性的表达产物，将提取的包涵体充分洗涤，用含有谷胱甘肽氧化还原系统的复性缓冲液处理，促进蛋白正确折叠复性。由于表达的融合蛋白含有His-tag，复性后的蛋白质通过Ni-IDA亲和重力柱分离纯化，在500mmol/L咪唑浓度下洗脱得到单一蛋白条带，纯化的蛋白浓度在0.4-0.7mg/mL。纯化的蛋白质经肠激酶酶切后，SDS-PAGE电泳出现两个蛋白条带，其中一个与片球菌素分子量相近，另一个与硫氧还蛋白的分子量相近。
纯化的蛋白质经肠激酶酶切后，牛津杯法测定酶切产物的抑菌活性，发现对单核细胞增多症李氏杆菌具有抑制作用，表明本试验建立的pedA基因大肠杆菌表达系统可以表达片球菌素融合蛋白，经包涵体复性、融合蛋白酶切后得到了有活性的片球菌素产物。对得到的基因工程片球菌素，用牛津杯法测定其抑菌谱，除对单核细胞增多症李氏杆菌有抑菌作用外，对植物乳杆菌也有抑制作用，但对金黄色葡萄球菌、沙门氏菌、大肠杆菌O157无抑制作用。
As one of natural biopreservatives, bacteriocin plays important role in improvingfood safety and extending shelf-life. This research subject made use of geneengineering technology to clone the pediocin pedA gene from Pediococcusacidilactici and construct pediocin pedA gene expression system in Escherichia coliin order to solve the low bacteriocin production problem in wild strain and realizeexpected highly efficient expression of pediocin.
The total DNA of P. acidilactici was used as the template to amplify thestructural gene pedA in the method of polymerase chain reaction (PCR). Theamplified pediocin structural gene pedA was ligased with pMD18-T vector beforetransformed E. coli DH5α competent cells. The pMD18-T recombinant plasmidcontaining pedA gene was extracted and digested by Bgl II和Xhol I restrictionenzymes and then ligased with the expression vector pET32a(+) which was digestedwith the same restriction enzymes. The ligation mixture was used to transform E. coliDH5α competent cells and further E. coli BL21(DE3) competent cells. Therecombinant strains were screened by PCR method and one was selected to extractrecombinant plasmid for DNA sequencing of the pedA gene. The result testified thatthe recombinant plasmid contained the pedA gene which was100%homologous tothe pedA gene published in the NCBI (GenBank AY083244). This recombinant strainwas induced with1mmol/L IPTG at37℃for4h and efficiently expressed22kDaTrx-Ped Afusion protein.
The culture of the recombinant was sonicated and centrifuged. Both thesupernatant and the pellet were analized by SDS-PAGE. Since the result showed thatthe expected fusion protein was in the pellet, the protein was expressed as inclusionbody. To obtain active expression product, the extracted inclusion body was washedadequately and treated with the renaturation buffer containing GSSG-GSH system forthe correct refolding of the protein. The refolding protein took advantage of its ownHis-tag and was separated and purified by adding to the Ni-IDA agarose resin column.A single protein band was observed in500mmol/L imidazol elute. The purified fusionprotein has the concentration of0.4-0.7mg/mL. Purified fusion protein was treatedwith enterokinase and two protein bands appeared. One of them corresponded withthe predicted molecular mass of pedA gene; the other corresponded with the predicted molecular mass of thioredoxin.
Purified fusion protein was treated with enterokinase and the well test revealedthat the cleavage product had antimicrobial activity to Listeria monocytogenes whichproved that the constructed pediocin pedA gene expression system in E. coli couldexpress pediocin fusion protein and obtain biological active pediocin product afterinclusion body renaturation and enterokinase treatment of the fusion protein. The welltest evaluated the inhibitory spectrum of the genetic engineering pediocin. The resultsindicated that the antimicrobial activity on and Lactobacillus plantarum as well as L.monocytogenes was found clearly and the antimicrobial activity againstStaphylococcus aureus, Salmonella typhi and E. coli O157was not found.
以乳酸片球菌基因组为模板，采用PCR法，扩增出了片球菌素的结构基因pedA。将扩增的pedA基因连接到pMD18-T载体，转化至大肠杆菌DH5α。提取含有pedA基因的pMD18-T重组质粒，Bgl II和Xhol I双酶切获得pedA基因，与经同样双酶切后的表达质粒pET32a(+)连接，先后转化至大肠杆菌DH5α和大肠杆菌BL21(DE3)，用PCR法筛选阳性克隆菌株后，选取其中的一株阳性克隆菌株，提取重组质粒，进行pedA基因测序，发现该重组质粒含有pedA基因，其核苷酸序列与NCBI公布的片球菌素pedA基因（GenBank AY083244）同源性为100%。该菌株在含有1mmol/L IPTG的培养基中，37℃培养4小时后，高效表达了预期大小的22kDa硫氧还蛋白和片球菌素的融合蛋白。
重组菌培养物经超声破碎后离心，分别取上清和沉淀物用SDS-PAGE进行分析，由于在沉淀物SDS-PAGE电泳结果中有预期大小的融合蛋白条带，表明表达的蛋白为包涵体。为获得有活性的表达产物，将提取的包涵体充分洗涤，用含有谷胱甘肽氧化还原系统的复性缓冲液处理，促进蛋白正确折叠复性。由于表达的融合蛋白含有His-tag，复性后的蛋白质通过Ni-IDA亲和重力柱分离纯化，在500mmol/L咪唑浓度下洗脱得到单一蛋白条带，纯化的蛋白浓度在0.4-0.7mg/mL。纯化的蛋白质经肠激酶酶切后，SDS-PAGE电泳出现两个蛋白条带，其中一个与片球菌素分子量相近，另一个与硫氧还蛋白的分子量相近。
纯化的蛋白质经肠激酶酶切后，牛津杯法测定酶切产物的抑菌活性，发现对单核细胞增多症李氏杆菌具有抑制作用，表明本试验建立的pedA基因大肠杆菌表达系统可以表达片球菌素融合蛋白，经包涵体复性、融合蛋白酶切后得到了有活性的片球菌素产物。对得到的基因工程片球菌素，用牛津杯法测定其抑菌谱，除对单核细胞增多症李氏杆菌有抑菌作用外，对植物乳杆菌也有抑制作用，但对金黄色葡萄球菌、沙门氏菌、大肠杆菌O157无抑制作用。
As one of natural biopreservatives, bacteriocin plays important role in improvingfood safety and extending shelf-life. This research subject made use of geneengineering technology to clone the pediocin pedA gene from Pediococcusacidilactici and construct pediocin pedA gene expression system in Escherichia coliin order to solve the low bacteriocin production problem in wild strain and realizeexpected highly efficient expression of pediocin.
The total DNA of P. acidilactici was used as the template to amplify thestructural gene pedA in the method of polymerase chain reaction (PCR). Theamplified pediocin structural gene pedA was ligased with pMD18-T vector beforetransformed E. coli DH5α competent cells. The pMD18-T recombinant plasmidcontaining pedA gene was extracted and digested by Bgl II和Xhol I restrictionenzymes and then ligased with the expression vector pET32a(+) which was digestedwith the same restriction enzymes. The ligation mixture was used to transform E. coliDH5α competent cells and further E. coli BL21(DE3) competent cells. Therecombinant strains were screened by PCR method and one was selected to extractrecombinant plasmid for DNA sequencing of the pedA gene. The result testified thatthe recombinant plasmid contained the pedA gene which was100%homologous tothe pedA gene published in the NCBI (GenBank AY083244). This recombinant strainwas induced with1mmol/L IPTG at37℃for4h and efficiently expressed22kDaTrx-Ped Afusion protein.
The culture of the recombinant was sonicated and centrifuged. Both thesupernatant and the pellet were analized by SDS-PAGE. Since the result showed thatthe expected fusion protein was in the pellet, the protein was expressed as inclusionbody. To obtain active expression product, the extracted inclusion body was washedadequately and treated with the renaturation buffer containing GSSG-GSH system forthe correct refolding of the protein. The refolding protein took advantage of its ownHis-tag and was separated and purified by adding to the Ni-IDA agarose resin column.A single protein band was observed in500mmol/L imidazol elute. The purified fusionprotein has the concentration of0.4-0.7mg/mL. Purified fusion protein was treatedwith enterokinase and two protein bands appeared. One of them corresponded withthe predicted molecular mass of pedA gene; the other corresponded with the predicted molecular mass of thioredoxin.
Purified fusion protein was treated with enterokinase and the well test revealedthat the cleavage product had antimicrobial activity to Listeria monocytogenes whichproved that the constructed pediocin pedA gene expression system in E. coli couldexpress pediocin fusion protein and obtain biological active pediocin product afterinclusion body renaturation and enterokinase treatment of the fusion protein. The welltest evaluated the inhibitory spectrum of the genetic engineering pediocin. The resultsindicated that the antimicrobial activity on and Lactobacillus plantarum as well as L.monocytogenes was found clearly and the antimicrobial activity againstStaphylococcus aureus, Salmonella typhi and E. coli O157was not found.
引文
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