猪链球菌Ⅱ型MRP基因单价候选疫苗株的研究
摘要
猪链球菌Ⅱ型(Streptococcus suis serotye2, SS2)是当前最具致病性和危害性的猪链球菌类型之一,可引起多种人畜共患疾病,甚至死亡。基因工程疫苗同传统灭活疫苗相比,更为安全有效、成本低廉,利于大规模推广。本研究采用已报道的SS2候选中和性抗原——溶菌酶释放蛋白(Muramidase released protein, MRP)和具有免疫佐剂功能的猪源肠毒素大肠杆菌不耐热肠毒素elt基因,并依靠宿主-质粒平衡致死系统,尝试构建无抗生素抗性基因,更为安全稳定的SS2MRP单价候选疫苗株,为SS2防治探索有效方法。本研究共分三个部分。
(一)共表达mrp、asd沙门氏菌的构建
在无氧条件下采用肉汤培养基培养SS2四川分离株,提取所得菌株的基因组DNA,以此为模板经PCR扩增mrp全长基因,获得的PCR产物与pMD-18T载体连接,转入DH5a中并提取质粒。通过PCR检测mrp是否已插入,Kpn Ⅰ酶切检测mrp插入方向是否正确。选择与LacZ启动子方向相同的重组质粒,再经Pvu Ⅰ酶切,回收大小为5889bp的产物片段,使用T4DNA Polymerase补平末端。同时,以含有天冬氨酸半醛脱氢酶(Aspartic semialdehyde dehydrogenase, ASD)基因的质粒pZLZ1为模板扩增asd全长基因,将其与补平末端的重组质粒连接,插入到重组质粒ampr基因内部,将其电转入asd基因缺陷型大肠杆菌X6097(asd)中,经PCR鉴定构建成功后,将重组质粒转入中间沙门氏菌X3730(asd)中,最后转入asd基因缺陷型的沙门氏菌X4072(asd)中,形成依赖于asd基因的质粒—染色体平衡致死系统。诱导表达并电泳检测MRP蛋白显示重组菌株稳定表达MRP。
(二)构建elt基因表达质粒
以含有猪源不耐热肠毒素基因elt的质粒pMM085为模板扩增elt基因,并通过设计特异引物在基因两端分别引入MluI和ApaI位点。MluI、Apal双酶切PCR产物与pGEX6-P-1质粒,分别回收1213bp片段和4771bp片段。连接两段回收产物,转入DH5a中。经PCR检测重组质粒构建成功。
(三)构建mrp、eltB共表达质粒
按照第一部分的方法获得SS2基因组DNA,以此为模板扩增引入了MlsI和SalI位点的mrp基因。将扩增产物克隆至T载体上,命名为pTM。MlsI、SalI双酶切质粒pTM和pGEX6-P-1,分别回收大小为3991bp和4481bp的片段并将两者连接,构建为重组质粒pZM01。与此同时,设计引物以质粒pMM085为模板扩增猪源不耐热肠毒素的抗原表位及其B亚基的基因eltB并加入酶切位点MluI和Apal。双酶切PCR产物与pZM01,回收407bp片段和8286bp片段。连接两者构建成为pZM02,以该重组质粒为模板扩增mrp基因和eltB基因,得到的片段大小与预测相符。
以上实验为研制可用于实际防疫工作的安全高效的重组疫苗打下了理论和实践基础。
Streptococcus suis H is one of the most pathogenic and danger of streptococcus suis typese. It could lead to a series of inflammation which lead to death. Genetic engineering vaccine is most safe, low cost and effective method, easy to promote on a large scale in means of many epidemic preventions to streptococcus suis2. Muramidase released protein (MRP) is one of the first confirmed SS2's pathogenic factors. MRP has the most obvious immunogenicity. This reserch use this important virulence factor as immune original. And this reserch also study on enterotoxigenic
Escherichia coli heatliable toxin gene (elt). At the same time a plasmid-chromosome balanced-lethal system was used to conduct a SS2's MRP candidate recombinant vaccine strains without antibiotics resistance genes. This experiment is divided into three parts.
Firstly using broth medium in anaerobic conditions cultivture SS2sichuan separation strain. Isolate the muramidase released protein gene of Streptococcus suis serotypes2by PCR with the Streptococcus suis serotypes2strain from Sichuan in China, Ligate it to the pMD18-T vector. Kpn I digest recombinant vector to make sure the insert direction is correct. After choose the right insert plasmid using Pvu I digest it and Recover the5889bp fragment. At the same time, amplify asd gene from plasmid pZLZl. The insert it to recombinant vector and the antibiotic resistance gene ampr of the vector was destroyed by insertion of as a gene. Then the recombinant vector was successively transformed into E.coli X6097, Salmonella X3O37(asd+) and Salmonella X4072(asd). A plasmid-chromosome balanced-lethal system was conducted. The SDS-PAGE results indicated MRP was expressed by Salmonella X4072.
The second part is cloning the complete elt gene from plasmid pMM085by designing specific primers with new restriction sites. Then double digest it and pGEX6-P-1expression vector. Let1231bp and4771bp fragments in one by a ligase. The PCR detection indicated that restructuring plasmid was successfully built.
The last part is construct a recombinant expression plasmid ofmrp and eltB gene. Get the mrp gene from SS2train adding two restriction site MlsI and Sall. Product was be directly amplified to T vector, name this restructuring plasmid pTM. Then double digest pTM and pGEX6-P-1expression vector by MlsI and SalI.Recovery1231bp and4771bp fragments and link them, name the new restructuring plasmid as pZMO1. At the same time, clone the complete eltB gene from plasmid pMM085by designing specific primers with new restriction sitesMluI and ApaI. Recovey and let the407bp and8286bp fragments in one by a ligase, name the new restructuring plasmid as pZM02. The result of PCR mrp gene and eltB gene from pZM02indicated that recombinant plasmid was successfully built.
This research lays the foundation for further study.
(一)共表达mrp、asd沙门氏菌的构建
在无氧条件下采用肉汤培养基培养SS2四川分离株,提取所得菌株的基因组DNA,以此为模板经PCR扩增mrp全长基因,获得的PCR产物与pMD-18T载体连接,转入DH5a中并提取质粒。通过PCR检测mrp是否已插入,Kpn Ⅰ酶切检测mrp插入方向是否正确。选择与LacZ启动子方向相同的重组质粒,再经Pvu Ⅰ酶切,回收大小为5889bp的产物片段,使用T4DNA Polymerase补平末端。同时,以含有天冬氨酸半醛脱氢酶(Aspartic semialdehyde dehydrogenase, ASD)基因的质粒pZLZ1为模板扩增asd全长基因,将其与补平末端的重组质粒连接,插入到重组质粒ampr基因内部,将其电转入asd基因缺陷型大肠杆菌X6097(asd)中,经PCR鉴定构建成功后,将重组质粒转入中间沙门氏菌X3730(asd)中,最后转入asd基因缺陷型的沙门氏菌X4072(asd)中,形成依赖于asd基因的质粒—染色体平衡致死系统。诱导表达并电泳检测MRP蛋白显示重组菌株稳定表达MRP。
(二)构建elt基因表达质粒
以含有猪源不耐热肠毒素基因elt的质粒pMM085为模板扩增elt基因,并通过设计特异引物在基因两端分别引入MluI和ApaI位点。MluI、Apal双酶切PCR产物与pGEX6-P-1质粒,分别回收1213bp片段和4771bp片段。连接两段回收产物,转入DH5a中。经PCR检测重组质粒构建成功。
(三)构建mrp、eltB共表达质粒
按照第一部分的方法获得SS2基因组DNA,以此为模板扩增引入了MlsI和SalI位点的mrp基因。将扩增产物克隆至T载体上,命名为pTM。MlsI、SalI双酶切质粒pTM和pGEX6-P-1,分别回收大小为3991bp和4481bp的片段并将两者连接,构建为重组质粒pZM01。与此同时,设计引物以质粒pMM085为模板扩增猪源不耐热肠毒素的抗原表位及其B亚基的基因eltB并加入酶切位点MluI和Apal。双酶切PCR产物与pZM01,回收407bp片段和8286bp片段。连接两者构建成为pZM02,以该重组质粒为模板扩增mrp基因和eltB基因,得到的片段大小与预测相符。
以上实验为研制可用于实际防疫工作的安全高效的重组疫苗打下了理论和实践基础。
Streptococcus suis H is one of the most pathogenic and danger of streptococcus suis typese. It could lead to a series of inflammation which lead to death. Genetic engineering vaccine is most safe, low cost and effective method, easy to promote on a large scale in means of many epidemic preventions to streptococcus suis2. Muramidase released protein (MRP) is one of the first confirmed SS2's pathogenic factors. MRP has the most obvious immunogenicity. This reserch use this important virulence factor as immune original. And this reserch also study on enterotoxigenic
Escherichia coli heatliable toxin gene (elt). At the same time a plasmid-chromosome balanced-lethal system was used to conduct a SS2's MRP candidate recombinant vaccine strains without antibiotics resistance genes. This experiment is divided into three parts.
Firstly using broth medium in anaerobic conditions cultivture SS2sichuan separation strain. Isolate the muramidase released protein gene of Streptococcus suis serotypes2by PCR with the Streptococcus suis serotypes2strain from Sichuan in China, Ligate it to the pMD18-T vector. Kpn I digest recombinant vector to make sure the insert direction is correct. After choose the right insert plasmid using Pvu I digest it and Recover the5889bp fragment. At the same time, amplify asd gene from plasmid pZLZl. The insert it to recombinant vector and the antibiotic resistance gene ampr of the vector was destroyed by insertion of as a gene. Then the recombinant vector was successively transformed into E.coli X6097, Salmonella X3O37(asd+) and Salmonella X4072(asd). A plasmid-chromosome balanced-lethal system was conducted. The SDS-PAGE results indicated MRP was expressed by Salmonella X4072.
The second part is cloning the complete elt gene from plasmid pMM085by designing specific primers with new restriction sites. Then double digest it and pGEX6-P-1expression vector. Let1231bp and4771bp fragments in one by a ligase. The PCR detection indicated that restructuring plasmid was successfully built.
The last part is construct a recombinant expression plasmid ofmrp and eltB gene. Get the mrp gene from SS2train adding two restriction site MlsI and Sall. Product was be directly amplified to T vector, name this restructuring plasmid pTM. Then double digest pTM and pGEX6-P-1expression vector by MlsI and SalI.Recovery1231bp and4771bp fragments and link them, name the new restructuring plasmid as pZMO1. At the same time, clone the complete eltB gene from plasmid pMM085by designing specific primers with new restriction sitesMluI and ApaI. Recovey and let the407bp and8286bp fragments in one by a ligase, name the new restructuring plasmid as pZM02. The result of PCR mrp gene and eltB gene from pZM02indicated that recombinant plasmid was successfully built.
This research lays the foundation for further study.
引文
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[4]陈卫.猪链球菌3种主要毒力因子多重PCR快速检测方法的建立及其初步应用[D].南京农业大学硕士论文,2008.
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