肺炎链球菌表面蛋白A基因和溶血素基因的融合及高效表达
发布时间：2018-11-11 14:21
【摘要】： 由于肺炎链球菌对人类危害大且耐药菌株不断增多,肺炎链球菌疫苗的预防作用越来越重要。肺炎链球菌表面蛋白A(PspA)和溶血素(Ply)已被确定为肺炎链球菌疫苗的候选因子,但它们免疫保护作用的侧重点不同。PspA的免疫保护作用主要是抗S.pn的定殖,Ply是S.pn细胞内毒素,其免疫保护性必须在S.pn自溶后才能发生。天然的Ply在小鼠体内具有免疫保护作用,但是它具有细胞毒性,作为人类疫苗是不合适的。对Ply的细胞毒作用和补体激活必需的基因片段△A146R147进行突变,制成具有免疫原性但对红细胞和有核细胞没有毒性作用的“肺炎链球菌溶血素”,用其免疫小鼠后发现能使细胞毒性降低99.5%以上,可作为载体蛋白广泛的应用于融合疫苗的研制。本研究拟将二者的优点结合起来,通过串联重组表达技术将PspA与Ply进行融合,构建了含有两种基因的融合表达质粒并进行了原核表达与鉴定,以便同时研究两种候选因子在S.pn蛋白疫苗研制中的作用。 后续实验中,将通过动物实验和体外细胞粘附抑制试验对重组融合蛋白的生物学活性进行鉴定,观察融合蛋白对动物的免疫保护作用及对肺炎链球菌粘附宿主细胞的抑制作用,从而探讨该融合蛋白在研制肺炎链球菌多基因亚单位疫苗中的潜在价值。 目的 利用基因定点突变技术扩增Ply突变基因△Ply,基因重组技术构建△Ply与PspA基因的串联融合表达蛋白PspA-△Ply 方法 1.根据肺炎链球菌TIGR4型表面蛋白A和溶血素基因序列设计引物,定点突变法扩增Ply突变基因,PCR法扩增PspA基因。 2.运用基因重组技术构建pET32a-PspA-ΔPly表达载体,双酶切与测序验证重组质粒。 3.重组质粒转化入大肠杆菌BL21(DE3),经不同时间、不同温度,不同浓度IPTG诱导表达融合蛋白,Western Blot鉴定表达产物 结果 1.采用重叠PCR的方法成功扩增出Ply突变体基因片段△Ply,将其与载体连接后得到重组质粒pET32a-ΔPly,经双酶切和测序分析证实重组质粒克隆成功。 2.用普通PCR的方法扩增出与预期大小一致的PspA目的基因片段。目的基因与载体pET32a-ΔPly连接后的重组质粒PET32a-PspA-ΔPly,经双酶切鉴定及测序分析证实重组质粒克隆成功。 3.SDS-PAGE检测融合蛋白的分子量大小以及表达量的多少。Western blot验证融合蛋白的表达。 结论 成功实现Ply基因的定点突变,构建PspA-△Ply融合蛋白串联表达载体,并对该蛋白进行鉴定和分析,为研究新型抗S.pn蛋白疫苗奠定基础。
[Abstract]:The preventive effect of Streptococcus pneumoniae vaccine is becoming more and more important because of its great harm to human beings and the increasing number of drug-resistant strains. Streptococcus pneumoniae surface protein A (PspA) and hemolysin (Ply) have been identified as candidate factors for Streptococcus pneumoniae vaccine, but the focus of their immune protection is different. Ply is endotoxin in S.pn cells, and its immune protection must occur after autolysis of S.pn. Natural Ply has immune protection in mice, but it is not suitable as a human vaccine because of its cytotoxicity. By mutating the gene fragment A146R147 necessary for cytotoxicity and complement activation of Ply, Streptococcus pneumoniae hemolysin, which has immunogenicity but has no toxic effect on erythrocytes and nucleated cells, was made. It was found that it could reduce cytotoxicity by more than 99.5% after immunizing mice, and could be widely used as carrier protein in the development of fusion vaccine. In this study, the advantages of the two methods were combined, PspA and Ply were fused by tandem recombination expression technique, and the fusion expression plasmids containing two genes were constructed and expressed and identified in prokaryotic. In order to study the role of two candidate factors in the development of S.pn protein vaccine. In the following experiments, the biological activity of the recombinant fusion protein was identified by animal experiment and in vitro cell adhesion inhibition test, and the immunoprotective effect of the fusion protein on animal and the inhibitory effect on the adhesion of Streptococcus pneumoniae to host cells were observed. To explore the potential value of the fusion protein in the development of Streptococcus pneumoniae multigene subunit vaccine. Objective to construct the fusion protein PspA- Ply of Ply and PspA gene by amplification of Ply, gene of Ply mutation by site-directed mutation. According to the sequence of TIGR4 type surface protein A and hemolysin gene of Streptococcus pneumoniae, primers were designed to amplify Ply mutant gene by site-directed mutation and PspA gene by PCR method. 2. PET32a-PspA- 螖 Ply expression vector was constructed by gene recombination technique, and the recombinant plasmid was confirmed by double enzyme digestion and sequencing. 3. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and the expression product was identified by different time, different temperature and different concentration of IPTG induced expression of the fusion protein, Western Blot. The recombinant plasmid pET32a- 螖 Ply, was successfully cloned by double enzyme digestion and sequencing analysis. The recombinant plasmid pET32a- 螖 Ply, was successfully amplified by the method of overlapping PCR. The Ply gene fragment Ply, was ligated with the vector and the recombinant plasmid pET32a- 螖 Ply, was cloned successfully. 2. The target gene fragment of PspA was amplified by ordinary PCR. Objective the recombinant plasmid PET32a-PspA- 螖 Ply, which was ligated with the vector pET32a- 螖 Ply, was identified by double enzyme digestion and sequenced to confirm the successful cloning of the recombinant plasmid. The molecular weight of the fusion protein and the amount of. Western blot were detected by 3.SDS-PAGE to verify the expression of the fusion protein. Conclusion Site-directed mutation of Ply gene was successfully realized, PspA- Ply fusion protein tandem expression vector was constructed, and the protein was identified and analyzed, which laid a foundation for the study of novel anti-S.pn protein vaccine.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2325096
[Abstract]:The preventive effect of Streptococcus pneumoniae vaccine is becoming more and more important because of its great harm to human beings and the increasing number of drug-resistant strains. Streptococcus pneumoniae surface protein A (PspA) and hemolysin (Ply) have been identified as candidate factors for Streptococcus pneumoniae vaccine, but the focus of their immune protection is different. Ply is endotoxin in S.pn cells, and its immune protection must occur after autolysis of S.pn. Natural Ply has immune protection in mice, but it is not suitable as a human vaccine because of its cytotoxicity. By mutating the gene fragment A146R147 necessary for cytotoxicity and complement activation of Ply, Streptococcus pneumoniae hemolysin, which has immunogenicity but has no toxic effect on erythrocytes and nucleated cells, was made. It was found that it could reduce cytotoxicity by more than 99.5% after immunizing mice, and could be widely used as carrier protein in the development of fusion vaccine. In this study, the advantages of the two methods were combined, PspA and Ply were fused by tandem recombination expression technique, and the fusion expression plasmids containing two genes were constructed and expressed and identified in prokaryotic. In order to study the role of two candidate factors in the development of S.pn protein vaccine. In the following experiments, the biological activity of the recombinant fusion protein was identified by animal experiment and in vitro cell adhesion inhibition test, and the immunoprotective effect of the fusion protein on animal and the inhibitory effect on the adhesion of Streptococcus pneumoniae to host cells were observed. To explore the potential value of the fusion protein in the development of Streptococcus pneumoniae multigene subunit vaccine. Objective to construct the fusion protein PspA- Ply of Ply and PspA gene by amplification of Ply, gene of Ply mutation by site-directed mutation. According to the sequence of TIGR4 type surface protein A and hemolysin gene of Streptococcus pneumoniae, primers were designed to amplify Ply mutant gene by site-directed mutation and PspA gene by PCR method. 2. PET32a-PspA- 螖 Ply expression vector was constructed by gene recombination technique, and the recombinant plasmid was confirmed by double enzyme digestion and sequencing. 3. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and the expression product was identified by different time, different temperature and different concentration of IPTG induced expression of the fusion protein, Western Blot. The recombinant plasmid pET32a- 螖 Ply, was successfully cloned by double enzyme digestion and sequencing analysis. The recombinant plasmid pET32a- 螖 Ply, was successfully amplified by the method of overlapping PCR. The Ply gene fragment Ply, was ligated with the vector and the recombinant plasmid pET32a- 螖 Ply, was cloned successfully. 2. The target gene fragment of PspA was amplified by ordinary PCR. Objective the recombinant plasmid PET32a-PspA- 螖 Ply, which was ligated with the vector pET32a- 螖 Ply, was identified by double enzyme digestion and sequenced to confirm the successful cloning of the recombinant plasmid. The molecular weight of the fusion protein and the amount of. Western blot were detected by 3.SDS-PAGE to verify the expression of the fusion protein. Conclusion Site-directed mutation of Ply gene was successfully realized, PspA- Ply fusion protein tandem expression vector was constructed, and the protein was identified and analyzed, which laid a foundation for the study of novel anti-S.pn protein vaccine.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 胥文春;曹炬;许颂霄;罗进勇;朱旦;尹一兵;;PspA免疫小鼠抗肺炎链球菌侵袭性感染研究[J];第四军医大学学报;2007年09期
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