Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange
Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange
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DOI：
10.1186/1471-2180-12-61
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发表时间：
2012-04-23
期刊：
影响因子：
4.2
通讯作者：
Panbangred, Watanalai
中科院分区：
文献类型：
--
作者：
Buasri, Wasin;Impoolsup, Attawut;Panbangred, Watanalai
背景资料：与原始的灭活全细胞疫苗相比，针对百日咳杆菌引起的百日咳的无细胞百日咳疫苗的安全性大大提高。无细胞百日咳疫苗的主要抗原百日咳毒素（PT）必须经过化学灭活才能获得相应的类毒素（PTd）。然而，该过程导致抗原的广泛变性。含有PTd或含有灭活S1的重组PT（rPT）、丝状血凝素（FHA）和百日咳毒素（PRN）的无细胞百日咳疫苗的开发表明，PRN的产量有限，而FHA产量过剩。为了提高抗原产量和工艺经济性，我们已经构建了菌株的百日咳博德特氏菌，产生增强水平的rPT和PRN。结果：三个重组菌株的百日咳博德特氏菌获得了同源重组使用等位基因交换载体，pSS 4245。在第一个构建体中，编码PT亚基S1的片段被两个突变（R9 K和E129 G）取代，从而去除PT毒性，获得Bp-WWC菌株。在第二个构建体中，将含有上述突变的PT结构基因的整个簇的第二个拷贝插入Bp-WWC染色体的其他位置，并获得Bp-WWD菌株。与Bp-WWC（2.61 +/- 0.16 μ g/mL）和野生型菌株（2.2 μ g/mL）相比，该菌株产生的rPT量增加（3.77 +/- 0.53 μ g/mL）。在第三构建体中，将prn基因的第二拷贝插入Bp-WWD的染色体中以获得Bp-WWE。菌株Bp-WWE在细胞提取物中产生4.18 +/-1.02 μ g/mL的PRN，其比Bp-WWC（2.48 +/-0.10 μ g/mL）和Bp-WWD（2.31 +/-0.17 μ g/mL）高约两倍。从Bp-WWD纯化的PTd在0.8-1.6 μ g/孔对中国仓鼠卵巢（CHO）细胞没有显示任何毒性，而从WT纯化的PT在2.6 μ g/孔时表现出细胞聚集终点。结论：我们构建了表达增加量的抗原、rPT或rPT和PRN的百日咳博德特氏菌菌株。第三种抗原FHA的表达没有变化（总是过量）。这些菌株将用于生产负担得起的无细胞百日咳疫苗。
Background: Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN.Results: Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 +/- 0.53 mu g/mL) compared to Bp-WWC (2.61 +/- 0.16 mu g/mL) and wild type strain (2.2 mu g/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 +/- 1.02 mu g/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 +/- 0.10 mu g/mL) and Bp-WWD (2.31 +/- 0.17 mu g/mL). Purified PTd from Bp-WWD at 0.8-1.6 mu g/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well.Conclusions: We have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.