肺炎链球菌表面粘附素A的原核表达和免疫保护性的初步研究
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摘要
目的:肺炎链球菌(Streptococcus pneumoniae,S.P)广泛分布于自然界,是一种条件致病菌,定植在40%健康人群鼻咽部,可以引起肺炎、中耳炎、菌血症、脑膜炎等疾病。据统计在发展中国家,每年大约有500万5岁以下的儿童死于肺炎链球菌引起的肺炎及脑膜炎。随着抗生素的广泛使用,肺炎链球菌对青霉素耐药现象日益严重,且呈多重耐药,严重影响治疗效果,因此应用疫苗进行有效的预防显得尤为重要。肺炎链球菌有90多个血清型,其中近30个血清型对人有致病性,为细菌性肺炎的主要病原体。目前使用的疫苗多为型特异性多糖疫苗,难以覆盖所有血清型,且对2岁以下婴幼儿及老年人的保护效果不理想。因此,寻找新的候选疫苗是有效预防细菌性肺炎的关键。
肺炎链球菌表面黏附素A(PsaA)为肺炎链球菌的黏附分子,是该菌所有血清型共有的一种遗传保守的、种特异性的表面蛋白抗原。PsaA在肺炎链球菌黏附呼吸道粘膜过程中发挥关键作用,是主要的毒力因子之一。PsaA抗原决定簇全部或部分暴露于细胞表面,具有较好的免疫原性。因此,PsaA蛋白有可能作为肺炎链球菌候选蛋白疫苗的抗原,同时还有可能用于检测患者血清中相应的抗体,为肺炎链球菌感染的快速诊断提供依据。
本研究采取基因工程方法构建重组载体,经表达获得PsaA蛋白,并利用动物主动免疫来初步验证其保护作用,研究其作为疫苗抗原的可行性。
方法:1.psaA基因的克隆、表达:利用PCR技术从肺炎链球菌基因组中扩增psaA片段,克隆后构建在原核表达载体pET-32a中;经测序与GenBank数据库中肺炎链球菌全基因组的psaA片段进行比对,将含有目的基因片段的重组载体转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达,饱和硫酸铵纯化蛋白;应用SDS-PAGE电泳技术对表达产物进行分析,并用自制的小鼠抗血清进行Western blot鉴定。
2.主动免疫实验:将40只昆明小鼠随机分为2组(每组20只),分别为生理盐水对照组和PsaA实验组。实验组分别于0、14、21天经腹股沟皮下注射PsaA蛋白,剂量为10μg/只/次,对照组注射无菌生理盐水。免疫结束后第7天对两组小鼠经腹腔感染致死剂量肺炎链球菌,连续监测21天,进行生存时间分析。
结果:1.psaA基因的克隆、表达:经快速PCR鉴定、酶切鉴定和基因测序结果显示,肺炎链球菌psaA基因片段被正确克隆到pET-32a原核表达载体中,核苷酸序列长度为870bp,与GenBank中相应序列完全符合,无碱基突变;经IPTG诱导,重组质粒在BL21(DE3)菌中表达出了分子量约为57kD的Trx-PsaA融合蛋白(PsaA蛋白约为37KD,硫氧还原蛋白约20KD),表达的蛋白以可溶形式为主,表达量约占全菌总蛋白的60%,饱和硫酸铵纯化后纯度大于80%;重组蛋白能够与S.P免疫小鼠的抗血清发生结合反应。
2.重组蛋白PsaA的主动免疫实验结果:以PsaA蛋白免疫的小鼠半数生存时间与对照组相比明显延长(P<0.001)。
结论:1.成功构建了pET-32a-psaA重组表达载体。表达的重组蛋白PsaA具有良好的抗体结合活性。为肺炎链球菌感染的快速诊断抗原的制备提供实验依据。
2.PsaA蛋白能够保护昆明小鼠抵抗致死剂量肺炎链球菌的攻击,具有一定的免疫保护性,为肺炎链球菌蛋白疫苗的研制奠定了基础。
Objective: Streptococcus pneumoniae is a kind of conditioned pathogens to establish pharynx nasalis of 40% healthy crowd, and lead to pneumonia, otitis media, bacteremia, meningitis diseases and so on. It is a leading cause of morbidity and mortality in developed and developing countries. Each year S.P causes approximmately1.2 million deaths worldwide from pneumonia especially for old people and children. In developing countries, 5 million less 5 years old children died of pneumonia of Streptococcus pneumoniae every year. Streptococcus pneumoniae exten- sively distributes in nature, have 90 kinds of serotypes and patho- genicity for men mostly 30 serotypes in them, is one of main pathogens of bacterial pneumonia. At present capsular bolysaccharides vaccines are mostly used, but these difficultly contain all serotypes and fail to protect to infants and the elderly. Therefore, development of effective protein-based vaccines is pretty urgent.
PsaA is a kind of lipoprotein located in surface of Streptococcus pneu- moniae, and bring into main play during Streptococcus pneumoniae adhesiveing mucous membrane of respiratory tract, therefore PsaA concerns with invasion and virulence of Streptococcus pneumoniae. PsaA is a kind of surface protein antigen, which is possesed by all kinds of strains, genetic conservative and species characteristic. Coding open reading frame of psaA gene is 930 bp, which codes PsaA protein of 37KD. Spatial framework of mature PsaA is one a helix linking two (β/α)4 structural domains. We will construct custom-crafted gene, and obtain PsaA protein by means of expres-sion, and then identify its nature with Western blot for studying PsaA protein function in pathopoiesis of Streptococcus pneumoniae. Eventually we will immune animals with expressed protein, and investigate antibody.
Methods: 1 PsaA of Streptococcus pneumoniae gene and prokaryotic expression carrier pET32a were linked after cutting of two kinds of enzyme by gene recombination method in vitro, and were identificated by sequenc- ing. Eventually, the plasmids were transformed into Escherichia coli BL21 (DE3).The proteins were expressed with IPTG induction. Recombinant protein was detected with SDS-PAGE and purified with saturated ammon- ium sulfate. The antigenicity of proteins was confirmed with anti- Streptoc- occus pneumoniae mouse blood serum by Western blot.
2 Active immunization protection assay 40 kunming mice were divided 2 groups randomly: control group and PsaA proteins experimental group. PsaA proteins experimental group each mouse was received 10 ug of every time on day 0, 14, 21 by fold inguen hypodermic injection. Control group mice injected isotonic Na chloride. Each mouse was infected fatal dose S.P by abdominal cavity after immunity completion 7th day. These mice were then monitored for death over 21 days, and the median survival time of each mouse was recorded.
Results:
1.The results of tacho-PCR, restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene psaA had been cloned into the plasmid pET32a (+). The length of the psaA gene was 870bp. Compared to the sequence of GeneBank, no mutation was found. A recombinant protein about 57kD was expressed in BL21 (DE3) after induction of IPTG. SDS- PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 60% of total baterial protein and the purity of target protein was up to 80%. Western blot suggested that expressed protein was confirmed PsaA and displayed satisfactory antigenicity.
2.Active immunization protection assays In this experiment, the median survival times of PsaA proteins experimental group was signifi- cantly longer than control group(p<0.001).
Conclusion:
1.The psaA genes of S.P were cloned successfully into the plasmid pET-32a. Sequencing and sequencing analysis were done and no mutation was found. The expressed and purified recombinant proteins were proved with immunogenicity. The recombinant protein can be tentative antigen of rapid diagnosis for pneumonia of Streptococcus pneumoniae.
2.PsaA protein could elicit protection against fatal dose S.P aggression for Kunming mice. The recombinant protein can be candidate protein antigen of vaccines against S.P. It established foundation for investigation of protein vaccines of Streptococcus pneumoniae.
肺炎链球菌表面黏附素A(PsaA)为肺炎链球菌的黏附分子,是该菌所有血清型共有的一种遗传保守的、种特异性的表面蛋白抗原。PsaA在肺炎链球菌黏附呼吸道粘膜过程中发挥关键作用,是主要的毒力因子之一。PsaA抗原决定簇全部或部分暴露于细胞表面,具有较好的免疫原性。因此,PsaA蛋白有可能作为肺炎链球菌候选蛋白疫苗的抗原,同时还有可能用于检测患者血清中相应的抗体,为肺炎链球菌感染的快速诊断提供依据。
本研究采取基因工程方法构建重组载体,经表达获得PsaA蛋白,并利用动物主动免疫来初步验证其保护作用,研究其作为疫苗抗原的可行性。
方法:1.psaA基因的克隆、表达:利用PCR技术从肺炎链球菌基因组中扩增psaA片段,克隆后构建在原核表达载体pET-32a中;经测序与GenBank数据库中肺炎链球菌全基因组的psaA片段进行比对,将含有目的基因片段的重组载体转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达,饱和硫酸铵纯化蛋白;应用SDS-PAGE电泳技术对表达产物进行分析,并用自制的小鼠抗血清进行Western blot鉴定。
2.主动免疫实验:将40只昆明小鼠随机分为2组(每组20只),分别为生理盐水对照组和PsaA实验组。实验组分别于0、14、21天经腹股沟皮下注射PsaA蛋白,剂量为10μg/只/次,对照组注射无菌生理盐水。免疫结束后第7天对两组小鼠经腹腔感染致死剂量肺炎链球菌,连续监测21天,进行生存时间分析。
结果:1.psaA基因的克隆、表达:经快速PCR鉴定、酶切鉴定和基因测序结果显示,肺炎链球菌psaA基因片段被正确克隆到pET-32a原核表达载体中,核苷酸序列长度为870bp,与GenBank中相应序列完全符合,无碱基突变;经IPTG诱导,重组质粒在BL21(DE3)菌中表达出了分子量约为57kD的Trx-PsaA融合蛋白(PsaA蛋白约为37KD,硫氧还原蛋白约20KD),表达的蛋白以可溶形式为主,表达量约占全菌总蛋白的60%,饱和硫酸铵纯化后纯度大于80%;重组蛋白能够与S.P免疫小鼠的抗血清发生结合反应。
2.重组蛋白PsaA的主动免疫实验结果:以PsaA蛋白免疫的小鼠半数生存时间与对照组相比明显延长(P<0.001)。
结论:1.成功构建了pET-32a-psaA重组表达载体。表达的重组蛋白PsaA具有良好的抗体结合活性。为肺炎链球菌感染的快速诊断抗原的制备提供实验依据。
2.PsaA蛋白能够保护昆明小鼠抵抗致死剂量肺炎链球菌的攻击,具有一定的免疫保护性,为肺炎链球菌蛋白疫苗的研制奠定了基础。
Objective: Streptococcus pneumoniae is a kind of conditioned pathogens to establish pharynx nasalis of 40% healthy crowd, and lead to pneumonia, otitis media, bacteremia, meningitis diseases and so on. It is a leading cause of morbidity and mortality in developed and developing countries. Each year S.P causes approximmately1.2 million deaths worldwide from pneumonia especially for old people and children. In developing countries, 5 million less 5 years old children died of pneumonia of Streptococcus pneumoniae every year. Streptococcus pneumoniae exten- sively distributes in nature, have 90 kinds of serotypes and patho- genicity for men mostly 30 serotypes in them, is one of main pathogens of bacterial pneumonia. At present capsular bolysaccharides vaccines are mostly used, but these difficultly contain all serotypes and fail to protect to infants and the elderly. Therefore, development of effective protein-based vaccines is pretty urgent.
PsaA is a kind of lipoprotein located in surface of Streptococcus pneu- moniae, and bring into main play during Streptococcus pneumoniae adhesiveing mucous membrane of respiratory tract, therefore PsaA concerns with invasion and virulence of Streptococcus pneumoniae. PsaA is a kind of surface protein antigen, which is possesed by all kinds of strains, genetic conservative and species characteristic. Coding open reading frame of psaA gene is 930 bp, which codes PsaA protein of 37KD. Spatial framework of mature PsaA is one a helix linking two (β/α)4 structural domains. We will construct custom-crafted gene, and obtain PsaA protein by means of expres-sion, and then identify its nature with Western blot for studying PsaA protein function in pathopoiesis of Streptococcus pneumoniae. Eventually we will immune animals with expressed protein, and investigate antibody.
Methods: 1 PsaA of Streptococcus pneumoniae gene and prokaryotic expression carrier pET32a were linked after cutting of two kinds of enzyme by gene recombination method in vitro, and were identificated by sequenc- ing. Eventually, the plasmids were transformed into Escherichia coli BL21 (DE3).The proteins were expressed with IPTG induction. Recombinant protein was detected with SDS-PAGE and purified with saturated ammon- ium sulfate. The antigenicity of proteins was confirmed with anti- Streptoc- occus pneumoniae mouse blood serum by Western blot.
2 Active immunization protection assay 40 kunming mice were divided 2 groups randomly: control group and PsaA proteins experimental group. PsaA proteins experimental group each mouse was received 10 ug of every time on day 0, 14, 21 by fold inguen hypodermic injection. Control group mice injected isotonic Na chloride. Each mouse was infected fatal dose S.P by abdominal cavity after immunity completion 7th day. These mice were then monitored for death over 21 days, and the median survival time of each mouse was recorded.
Results:
1.The results of tacho-PCR, restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene psaA had been cloned into the plasmid pET32a (+). The length of the psaA gene was 870bp. Compared to the sequence of GeneBank, no mutation was found. A recombinant protein about 57kD was expressed in BL21 (DE3) after induction of IPTG. SDS- PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 60% of total baterial protein and the purity of target protein was up to 80%. Western blot suggested that expressed protein was confirmed PsaA and displayed satisfactory antigenicity.
2.Active immunization protection assays In this experiment, the median survival times of PsaA proteins experimental group was signifi- cantly longer than control group(p<0.001).
Conclusion:
1.The psaA genes of S.P were cloned successfully into the plasmid pET-32a. Sequencing and sequencing analysis were done and no mutation was found. The expressed and purified recombinant proteins were proved with immunogenicity. The recombinant protein can be tentative antigen of rapid diagnosis for pneumonia of Streptococcus pneumoniae.
2.PsaA protein could elicit protection against fatal dose S.P aggression for Kunming mice. The recombinant protein can be candidate protein antigen of vaccines against S.P. It established foundation for investigation of protein vaccines of Streptococcus pneumoniae.
引文
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23. Sampson JS, O,Connor SP,Stinson AR, et al. Cloning and nuclcotide sequence analysis of psaA,the Streptococcus pneumoniae gene encoding nucleotide a 37- kilodalton protein homologous to previously reported Streptococcus sp. adhesins.Infection and Immunity 1994; 62 (1):319-324.
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25. J.萨姆布鲁克,D.W.拉塞尔著(黄培堂等译)分子克隆实验指南[M].第3版,北京,科学出版社2002; 87-98.
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