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首页> 外文期刊>Infection and immunity >Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.
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Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.

机译：使用抗PAK菌毛和抗肽抗体对铜绿假单胞菌PAK和PAO菌毛蛋白的二硫键桥区域进行免疫学研究。

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Pseudomonas aeruginosa is an opportunistic pathogen that attaches to host cells via their pili. The pilus of P. aeruginosa PAK consists of a polymer of a single subunit, pilin, which is a 144-residue polypeptide. The C-terminal end of this protein is semiconserved in a number of strains and contains a disulfide bridge. We have synthesized the C-terminal peptide PAK (128-144)-OH in both its reduced and oxidized forms and the analog PAK(A-129) (128-144)-OH, in which cysteine-129 was substituted by alanine. These three peptides were used to immunize rabbits and prepare antipeptide antisera. It was found that antipeptide antisera to reduced peptide (17-R) and to oxidized peptide (17-O) bound to native PAK pili and cross-reacted with strain PAO pili in direct enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. However, the antiserum to the peptide immunogen PAK(A-129)(128-144)-OH, which does not have the ability to form the disulfide bridge, did not bind to either PAK or PAO pili. Competitive ELISA experiments with reduced and oxidized peptides of Ac-PAK(128-144)-OH showed that there was no difference in binding between the two peptides for 17-R or 17-O immunoglobulin G. When immunoglobulin G from native PAK antipilus antiserum was used in competitive or direct ELISA experiments, there was also no preference in binding to reduced or oxidized Ac-PAK(128-144)-OH or to PAK(A-129)(128-144)-OH. This result showed that the disulfide bridge in Pseudomonas pili is not critical to the immunogenicity of this region. However, the disulfide bridge is important in the immunogenicity of the C-terminal peptide when preparing antipeptide antisera that are cross-reactive with pili from different strains, since only the disulfide bridge peptide antisera cross-reacted well with the PAO pili as shown by competitive ELISA, suggesting that this region could be an important candidate for development of a synthetic vaccine.
机译：铜绿假单胞菌是通过其菌毛附着于宿主细胞的机会病原体。铜绿假单胞菌PAK的菌毛由单个亚基菌毛蛋白的聚合物组成，菌毛蛋白为144个残基的多肽。该蛋白的C末端在许多菌株中是半保守的，并包含一个二硫键。我们已经合成了还原和氧化形式的C末端肽PAK（128-144）-OH和类似物PAK（A-129）（128-144）-OH，其中半胱氨酸-129被丙氨酸取代。这三种肽用于免疫兔并制备抗肽抗血清。发现在直接酶联免疫吸附测定（ELISA）和免疫印迹实验中，还原肽（17-R）和氧化肽（17-O）的抗肽抗血清与天然PAK菌毛结合并与菌株PAO菌毛发生交叉反应。但是，针对肽免疫原PAK（A-129）（128-144）-OH的抗血清不具有形成二硫键的能力，因此它既不与PAK结合也不与PAO菌毛结合。用Ac-PAK（128-144）-OH还原肽和氧化肽进行的竞争ELISA实验表明，两种肽对17-R或17-O免疫球蛋白G的结合没有差异。当来自天然PAK抗菌抗血清的免疫球蛋白G时将其用于竞争性或直接ELISA实验中，也没有优先选择结合还原或氧化的Ac-PAK（128-144）-OH或PAK（A-129）（128-144）-OH。该结果表明，在假单胞菌菌毛中的二硫键对该区域的免疫原性不是关键的。但是，当制备与不同菌株的菌毛交叉反应的抗肽抗血清时，二硫键对C末端肽的免疫原性很重要，因为竞争法证明只有二硫桥肽抗血清与PAO菌毛交叉反应良好。 ELISA，表明该区域可能是合成疫苗开发的重要候选对象。

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