肺炎鏈球菌表面蛋白A基因和溶血素基因的融合及高效表達(dá)
發(fā)布時(shí)間：2018-11-11 14:21
【摘要】： 由于肺炎鏈球菌對(duì)人類危害大且耐藥菌株不斷增多,肺炎鏈球菌疫苗的預(yù)防作用越來越重要。肺炎鏈球菌表面蛋白A(PspA)和溶血素(Ply)已被確定為肺炎鏈球菌疫苗的候選因子,但它們免疫保護(hù)作用的側(cè)重點(diǎn)不同。PspA的免疫保護(hù)作用主要是抗S.pn的定殖,Ply是S.pn細(xì)胞內(nèi)毒素,其免疫保護(hù)性必須在S.pn自溶后才能發(fā)生。天然的Ply在小鼠體內(nèi)具有免疫保護(hù)作用,但是它具有細(xì)胞毒性,作為人類疫苗是不合適的。對(duì)Ply的細(xì)胞毒作用和補(bǔ)體激活必需的基因片段△A146R147進(jìn)行突變,制成具有免疫原性但對(duì)紅細(xì)胞和有核細(xì)胞沒有毒性作用的“肺炎鏈球菌溶血素”,用其免疫小鼠后發(fā)現(xiàn)能使細(xì)胞毒性降低99.5%以上,可作為載體蛋白廣泛的應(yīng)用于融合疫苗的研制。本研究擬將二者的優(yōu)點(diǎn)結(jié)合起來,通過串聯(lián)重組表達(dá)技術(shù)將PspA與Ply進(jìn)行融合,構(gòu)建了含有兩種基因的融合表達(dá)質(zhì)粒并進(jìn)行了原核表達(dá)與鑒定,以便同時(shí)研究?jī)煞N候選因子在S.pn蛋白疫苗研制中的作用。 后續(xù)實(shí)驗(yàn)中,將通過動(dòng)物實(shí)驗(yàn)和體外細(xì)胞粘附抑制試驗(yàn)對(duì)重組融合蛋白的生物學(xué)活性進(jìn)行鑒定,觀察融合蛋白對(duì)動(dòng)物的免疫保護(hù)作用及對(duì)肺炎鏈球菌粘附宿主細(xì)胞的抑制作用,從而探討該融合蛋白在研制肺炎鏈球菌多基因亞單位疫苗中的潛在價(jià)值。 目的 利用基因定點(diǎn)突變技術(shù)擴(kuò)增Ply突變基因△Ply,基因重組技術(shù)構(gòu)建△Ply與PspA基因的串聯(lián)融合表達(dá)蛋白PspA-△Ply 方法 1.根據(jù)肺炎鏈球菌TIGR4型表面蛋白A和溶血素基因序列設(shè)計(jì)引物,定點(diǎn)突變法擴(kuò)增Ply突變基因,PCR法擴(kuò)增PspA基因。 2.運(yùn)用基因重組技術(shù)構(gòu)建pET32a-PspA-ΔPly表達(dá)載體,雙酶切與測(cè)序驗(yàn)證重組質(zhì)粒。 3.重組質(zhì)粒轉(zhuǎn)化入大腸桿菌BL21(DE3),經(jīng)不同時(shí)間、不同溫度,不同濃度IPTG誘導(dǎo)表達(dá)融合蛋白,Western Blot鑒定表達(dá)產(chǎn)物 結(jié)果 1.采用重疊PCR的方法成功擴(kuò)增出Ply突變體基因片段△Ply,將其與載體連接后得到重組質(zhì)粒pET32a-ΔPly,經(jīng)雙酶切和測(cè)序分析證實(shí)重組質(zhì)�？寺〕晒Α� 2.用普通PCR的方法擴(kuò)增出與預(yù)期大小一致的PspA目的基因片段。目的基因與載體pET32a-ΔPly連接后的重組質(zhì)粒PET32a-PspA-ΔPly,經(jīng)雙酶切鑒定及測(cè)序分析證實(shí)重組質(zhì)�？寺〕晒Α� 3.SDS-PAGE檢測(cè)融合蛋白的分子量大小以及表達(dá)量的多少。Western blot驗(yàn)證融合蛋白的表達(dá)。 結(jié)論 成功實(shí)現(xiàn)Ply基因的定點(diǎn)突變,構(gòu)建PspA-△Ply融合蛋白串聯(lián)表達(dá)載體,并對(duì)該蛋白進(jìn)行鑒定和分析,為研究新型抗S.pn蛋白疫苗奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 胥文春;曹炬;許頌霄;羅進(jìn)勇;朱旦;尹一兵;;PspA免疫小鼠抗肺炎鏈球菌侵襲性感染研究[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2007年09期
，本文編號(hào)：2325096
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2325096.html
最近更新
教材專著
