Suvorov A, Ustinovitch I, Meringova L, Grabovskaya K, Leontieva G, Vorobieva E, Totolian A
Institute of Experimental Medicine, Academy of Medical Sciences, Pavlova 12,St. Petersburg 197376, Russia.
Indian J Med Res. 2004 May;119 Suppl:228-32.
BACKGROUND & OBJECTIVES: immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection.
Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies.
Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection.
INTERPRETATION & CONCLUSION: Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.
免疫功能低下的成年人。约50%的B群链球菌（GBS）菌株携带并表达能结合IgA的BAC抗原基因。编码BAC抗原的基因已被克隆和测序，但该蛋白上实际的IgA结合区域尚未被检测到。本研究的目的是定位Bac蛋白上的IgA结合区域，评估Bac蛋白区域之一MLKKIE在IgA结合中的作用，并研究基于Bac的重组蛋白产生抗GBS感染保护性抗体的能力。
通过PCR扩增bac基因片段，随后将PCR产物克隆到表达质粒中，产生基于β抗原C的重组蛋白。通过免疫沉淀和蛋白质印迹法检测重组肽与IgA的结合情况。将一种表达IgA结合的重组蛋白用作抗原免疫小鼠并进行GBS保护研究。
构建了几种bac基因构建体。它们结合IgA的能力差异很大，这取决于构建体的大小和片段在bac基因图谱上的位置。表达IgA结合的最小肽大小为14kD。MLKKIE区域的氨基酸取代促进了IgA结合能力。用基于重组Bac的肽免疫小鼠可诱导出现高亲和力水平的抗GBS抗体，提供针对GBS感染的保护。
基于Bac的重组肽的大小依赖性证明，有效的IgA结合需要结合IgA的蛋白进行特定折叠。MLKKIE区域不能被视为负责IgA结合的区域。产生高滴度和亲和力的针对基于Bac的重组肽的抗体，使这些蛋白成为开发抗GBS感染疫苗的有力候选者。
