缺铁胁迫对海豚链球菌生长的影响及铁转运蛋白基因（ftsABCD）的克隆与表达
摘要
海豚链球菌(Streptococcus iniae)是一类重要的鱼类病原菌,由该菌引起的鱼类海豚链球菌病对当今世界的水产养殖业造成了巨大的经济损失。为了能有效的控制海豚链球菌病,需要对海豚链球菌毒力因子进行深入研究,寻找有效的抗原。铁元素是一种生命必须的营养元素,众多研究表明铁元素作为一种重要的环境信号因子影响着细菌相关毒力基因的表达,另外,负责铁转运的复合体属于ABC转运体家族,该转运体上的铁元素受体结合蛋白具有一定的抗原性,能作为亚单位疫苗的候选者之一。基于上述原因,本文选择对海豚链球菌进行铁胁迫以及铁转运基因的研究,希望能初步揭示铁元素对海豚链球菌的重要作用,以及通过分子生物学方法找到海豚链球菌铁转运体并对受体蛋白的抗原性进行验证,为亚单位疫苗的研制打下基础。
本研究使用次氮基三乙酸钠盐(Nitrilotriacetic acid trisodium salt,NTA)作为铁螯合剂添加到脑心浸液培养基中人为制造缺铁培养环境,观察海豚链球菌的生长情况。结果表明:加入终浓度为5 mM NTA能够延缓海豚链球菌HD-1菌株对数生长期时间6 hr,且静止期细菌密度是0.67,要显著小于对照组0.77,且革兰氏染色发现细菌形成长链状;加入终浓度为15 mM NTA则完全抑制海豚链球菌HD-1生长,再加入100μM血红蛋白或者100mM氯化铁则细菌恢复生长。进一步分析不同条件下生长的海豚链球菌细胞组分(SDS-PAGE电泳分析),发现对照组与5mM NTA处理组相比,其全菌可溶蛋白、全菌不可溶蛋白、原生质体破碎蛋白、胞壁蛋白、原生质体不可溶蛋白、原生质体可溶蛋白等成分均有差异。使用CAS检测法未能检测出海豚链球菌HD-1产生siderophore蛋白。此结果表明海豚链球菌并不是通过分泌siderophore来摄取铁源,而是另有途径。
在对缺铁胁迫培养条件下海豚链球菌的各种表观特征进行了初步研究后,本研究利用海豚链球菌ATCC 9117菌株盲测序列数据库,与从NCBI中获得的酿脓链球菌FtsABCD铁转运蛋白序列进行tblastn,找到同源性较高的海豚链球菌相关基因,并命名为海豚链球菌ftsABCD。设计特异性引物扩增基因全长后发现ftsA基因全长783 bp,编码260个氨基酸; ftsB基因全长为927 bp,编码308个氨基酸; ftsC基因全长为1,029 bp,编码342个氨基酸; ftsD基因全长为999 bp,编码332个氨基酸。经过生物信息学分析预测海豚链球菌FtsABCD为一个ABC转运体(ABC transporter),其中FtsA基因为ATP水解酶,FtsB为底物结合蛋白,FtsC与FtsD均为通透酶。
通过克隆引入了BamH I、Xho I双酶切位点的ftsB成熟肽序列,构建了原核表达质粒,然后将质粒转入大肠杆菌BL21进行FtsB重组蛋白的表达。通过优化温度、时间与诱导剂IPTG浓度,最终确定了重组蛋白表达的最佳条件。FtsB重组蛋白分子量为49.6 kDa,由于其带有组氨酸标签,因此用镍柱对混合蛋白进行纯化,然后对纯化的重组蛋白进行脱盐,冷冻干燥浓缩。
将海豚链球菌HD-1通过腹腔注射到小鼠体内,进行人工感染,在感染后第10天取感染小鼠血清作为一抗,碱性磷酸酶标记的马抗鼠抗体为二抗,对电转到硝酸纤维素膜上的FtsB重组蛋白进行western blot检测。结果发现,一抗能特异性的检测到FtsB重组蛋白。这表明海豚链球菌HD-1在感染小鼠的过程中,ftsB基因转录并表达,且表达产物FtsB蛋白能被小鼠免疫系统识别为有效抗原并产生了相应的抗体。因此,FtsB能够作为亚单位疫苗有效抗原的候选之一。
Streptococcus iniae is one of the most important fish pathogens that caused millions of dollars economic damage every year. In order to control Streptococcus iniae effectively, studies that focused on the virulence factors are needed. In this study, we find that iron starvation caused by addition of NTA in the final concentration 5mM postpones the log phase of bacterial growth almost 6 hours, when examined under microscope the gram stained bacteria showed a long chain appearance and the protein profile changed a lot compared with the positive control. Siderophore could not be detected by the universal CAS assay. Using the protein sequences of Streptococcus pyogene ferrichrome transporter FtsABCD, we searched the raw genome sequence of Streptococcus iniae 9117 provided by the Baylor College of Medicine. Then we designed 2 pairs of nested PCR primers for each gene based on the conserved domains and cloned part sequences of the ftsABCD of Streptococcus iniae. The cloned sequences matched perfectly with the genome sequences of Streptococcus iniae, in this case we designed the ORF primers based on the ORF predictions of the sequences from the database. As a result, the length of ftsA is 783 bp, ftsB is 927 bp, ftsC is 1029 bp, ftsD is 999bp. The results of signal peptide and transmembrane prediction, and combined with the results of NCBI‘s Conserved Domains search indicated that FtsABCD of Streptococcus iniae constitutes an ABC transporter in which FtsA is ATP binding protein, FtsB is substrate binding protein, FtsC and FtsD are permeases. Next, the prokaryote expression system of FtsB was constructed using the PCR products of ftsB mature sequence and the plasmid pET-32a. Then the recombinate FtsB protein was pured by Ni+ affinity chromatography and lyophilized and stored at -80℃.In the serum of mouse that have been infected by Streptococcus iniae we detected antibody against the recombinate FtsB protein specifically by western blot.
In concluding, iron is an essential nutrient that Streptococcus iniae needs to survive. And the ftsABCD gene of Streptococcus iniae constitutes an ABC transporter, ftsB gene is translated during the infection of mouse by Streptococcus iniae. Finally, the product of ftsB has some kind of immunogenicity and could be used as the candidate of subunit vaccine.
本研究使用次氮基三乙酸钠盐(Nitrilotriacetic acid trisodium salt,NTA)作为铁螯合剂添加到脑心浸液培养基中人为制造缺铁培养环境,观察海豚链球菌的生长情况。结果表明:加入终浓度为5 mM NTA能够延缓海豚链球菌HD-1菌株对数生长期时间6 hr,且静止期细菌密度是0.67,要显著小于对照组0.77,且革兰氏染色发现细菌形成长链状;加入终浓度为15 mM NTA则完全抑制海豚链球菌HD-1生长,再加入100μM血红蛋白或者100mM氯化铁则细菌恢复生长。进一步分析不同条件下生长的海豚链球菌细胞组分(SDS-PAGE电泳分析),发现对照组与5mM NTA处理组相比,其全菌可溶蛋白、全菌不可溶蛋白、原生质体破碎蛋白、胞壁蛋白、原生质体不可溶蛋白、原生质体可溶蛋白等成分均有差异。使用CAS检测法未能检测出海豚链球菌HD-1产生siderophore蛋白。此结果表明海豚链球菌并不是通过分泌siderophore来摄取铁源,而是另有途径。
在对缺铁胁迫培养条件下海豚链球菌的各种表观特征进行了初步研究后,本研究利用海豚链球菌ATCC 9117菌株盲测序列数据库,与从NCBI中获得的酿脓链球菌FtsABCD铁转运蛋白序列进行tblastn,找到同源性较高的海豚链球菌相关基因,并命名为海豚链球菌ftsABCD。设计特异性引物扩增基因全长后发现ftsA基因全长783 bp,编码260个氨基酸; ftsB基因全长为927 bp,编码308个氨基酸; ftsC基因全长为1,029 bp,编码342个氨基酸; ftsD基因全长为999 bp,编码332个氨基酸。经过生物信息学分析预测海豚链球菌FtsABCD为一个ABC转运体(ABC transporter),其中FtsA基因为ATP水解酶,FtsB为底物结合蛋白,FtsC与FtsD均为通透酶。
通过克隆引入了BamH I、Xho I双酶切位点的ftsB成熟肽序列,构建了原核表达质粒,然后将质粒转入大肠杆菌BL21进行FtsB重组蛋白的表达。通过优化温度、时间与诱导剂IPTG浓度,最终确定了重组蛋白表达的最佳条件。FtsB重组蛋白分子量为49.6 kDa,由于其带有组氨酸标签,因此用镍柱对混合蛋白进行纯化,然后对纯化的重组蛋白进行脱盐,冷冻干燥浓缩。
将海豚链球菌HD-1通过腹腔注射到小鼠体内,进行人工感染,在感染后第10天取感染小鼠血清作为一抗,碱性磷酸酶标记的马抗鼠抗体为二抗,对电转到硝酸纤维素膜上的FtsB重组蛋白进行western blot检测。结果发现,一抗能特异性的检测到FtsB重组蛋白。这表明海豚链球菌HD-1在感染小鼠的过程中,ftsB基因转录并表达,且表达产物FtsB蛋白能被小鼠免疫系统识别为有效抗原并产生了相应的抗体。因此,FtsB能够作为亚单位疫苗有效抗原的候选之一。
Streptococcus iniae is one of the most important fish pathogens that caused millions of dollars economic damage every year. In order to control Streptococcus iniae effectively, studies that focused on the virulence factors are needed. In this study, we find that iron starvation caused by addition of NTA in the final concentration 5mM postpones the log phase of bacterial growth almost 6 hours, when examined under microscope the gram stained bacteria showed a long chain appearance and the protein profile changed a lot compared with the positive control. Siderophore could not be detected by the universal CAS assay. Using the protein sequences of Streptococcus pyogene ferrichrome transporter FtsABCD, we searched the raw genome sequence of Streptococcus iniae 9117 provided by the Baylor College of Medicine. Then we designed 2 pairs of nested PCR primers for each gene based on the conserved domains and cloned part sequences of the ftsABCD of Streptococcus iniae. The cloned sequences matched perfectly with the genome sequences of Streptococcus iniae, in this case we designed the ORF primers based on the ORF predictions of the sequences from the database. As a result, the length of ftsA is 783 bp, ftsB is 927 bp, ftsC is 1029 bp, ftsD is 999bp. The results of signal peptide and transmembrane prediction, and combined with the results of NCBI‘s Conserved Domains search indicated that FtsABCD of Streptococcus iniae constitutes an ABC transporter in which FtsA is ATP binding protein, FtsB is substrate binding protein, FtsC and FtsD are permeases. Next, the prokaryote expression system of FtsB was constructed using the PCR products of ftsB mature sequence and the plasmid pET-32a. Then the recombinate FtsB protein was pured by Ni+ affinity chromatography and lyophilized and stored at -80℃.In the serum of mouse that have been infected by Streptococcus iniae we detected antibody against the recombinate FtsB protein specifically by western blot.
In concluding, iron is an essential nutrient that Streptococcus iniae needs to survive. And the ftsABCD gene of Streptococcus iniae constitutes an ABC transporter, ftsB gene is translated during the infection of mouse by Streptococcus iniae. Finally, the product of ftsB has some kind of immunogenicity and could be used as the candidate of subunit vaccine.
引文
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[83] Blanca De Las Rivas, Jose′L. García, Rubens Lo′pez and Pedro García. Purifica-tion and Polar Localization of Pneumococcal LytB, a Putative Endo-N-Acetylg-lucosaminidase: the Chain-Dispersing Murein Hydrolase. J Bacteriology. 2002, 184(18): 4988-5000.
[84] Wai-Leung Ng, Krystyna M. Kazmierczak and Malcolm E. Winkler. Defective cell wall synthesis in Streptococcus pneumonia R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator. Molecular Microbiology. 2004, 53(4): 1161-1175.
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[86]沈智华,钱冬,许文军,顾金华,邵健忠。红拟石首鱼海豚链球菌分离、鉴定及致病性研究。水生生物学报.2005,29(6):678-683.
[87]周素明,李安兴,马跃,刘瑞明.养殖鱼类链球菌病病原的分离鉴定及其16S rDNA分析.中山大学学报(自然科学版).2007, 46(2).