Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.
Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.
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从牙龈卟啉单胞菌（拟杆菌）中产生和纯化重组菌丝蛋白 381。
DOI：
10.1128/iai.61.3.1040-1047.1993
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发表时间：
1993
影响因子：
3.1
通讯作者：
Flood,PM
中科院分区：
文献类型：
--
作者：
Washington,OR;Deslauriers,M;Stevens,DP;Lyford,LK;Haque,S;Yan,Y;Flood,PM
菌毛蛋白是人牙周病原菌牙龈卟啉单胞菌（拟杆菌）菌毛的主要亚基蛋白。我们在这里描述的重组菌毛蛋白（r-菌毛蛋白）分离牙龈卟啉单胞菌381的产生和初步表征。通过聚合酶链反应产生编码菌毛蛋白基因的DNA片段，并将其克隆到表达载体pET 11b中。将含有重组基因的质粒转染大肠杆菌。在平板上选择氨苄青霉素抗性的克隆，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）单独筛选用IPTG（异丙基-β-D-硫代半乳糖苷）活化后的蛋白质产生。一个克隆，OW0.2，产生大量的42 kDa的蛋白质诱导后，用IPTG。该克隆含有pET 11b质粒，该质粒具有与编码菌毛蛋白的基因具有序列同源性的1-kb插入物。来自克隆OW0.2的大部分重组蛋白被发现存在于细胞质中的包涵体内。将蛋白质聚集体溶解在8 M尿素中，SDS-PAGE分析显示两个主要蛋白质条带，一个在42 kDa，另一个在17 kDa。这两种蛋白质在0.15 M NaCl下从DEAE-Sepharose柱共洗脱，并在Western印迹（免疫印迹）中与菌毛蛋白的兔抗血清反应。通过使用连续洗脱电泳进行大小分级，获得在SDS-PAGE中在42 kDa处给出单一蛋白条带的制剂。来自用来自牙龈卟啉单胞菌的菌毛蛋白或r-菌毛蛋白免疫的动物的淋巴结细胞在体外回忆测定中显示对牙龈卟啉单胞菌菌毛蛋白和r-菌毛蛋白的抗原特异性增殖。因此，看来r-菌毛素在化学、抗原性和血清学上与从牙龈卟啉单胞菌381分离的菌毛素相同。
Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.