Investigation Of Protective Truncated Protein And Panning The Epitopes Of Fba Protein

Objective: Group A Streptococcus(GAS), a strong pathogenic bacteria which cause a variety of purulent inflammation, scarlet fever, erysipelas, neonatal sepsis, meningitis, puerperal fever, and allergic diseases, can host in human nasopharynx and the intestinal tract for a long term. Furthermore, infection of GAS still can't be cured and people may be infected repeatly. As other infective diseases, application of vaccine is an important strategy to interfere GAS invading cells and promote host's ability to clear Streptococcus. One of the major candidates is the surface M protein, which is a primary virulence factor of GAS, unfortunately, it is restricted as a vaccine because its multiple serotypes and cross-reactions with human tissue. The studies about vaccine of GAS involve F1 protein, C5 peptidase, Streptococcus sugar, exotoxin, etc. in the different laboratories around the world, but none of them is more significant than M protein.Terao Y. el at, using the genome sequence of S. pyogenes strain SF370, found a novel Fibronectin-binding protein (Fba) of GAS. The fba gene consisted 1068 nucleotides and encode a protein with 355 amino acids and molecular mass of 37.8KDa[1]. The whole protein contains the putative signal-peptide region, α-helical-coiled-coil region, proline-rich repeats region, C-terminal wall-anchored region. It exists in many serotypes such as M1, 2, 4, 9, 13, 22, 28, 44, 49, 60, 67, 75, 77, 79, 80, 82, 87 and 89 serotypes. Among different serotype, Fba protein shows a high homology. Experimental results showed that Fba located on the surface of GAS play an important role in the invasion of epithelial cells. FbaA binding to FH and FLH-1 has been found to promote GAS adhere, invade to epithelial cells and has the functions of opsonification and antiphagocytosis [2], which suggested that Fba plays a significant role in survival, virulence and pathogenicity of Streptococcus [3].The pre-study has showed that Fba protein has a similar ability with M protein to induce protective immune response, thus, Fba protein has a potential as a vaccine candidate against GAS infection.In the initial study, we known that Fba posses strong immunogenicity and could elicit immune protection to pretect animals from infection. In this research, animals were immuned with the truncated Fba proteins which were expressed successfully,and then challenged with GAS to further investigate protective antibodies induced by each truncated Fba protein and determine the domain that posses the best immunogenicity. In addition, polyclonal antibodies against Fba were prepared, purified and used to panning phage random peptide library (Ph ? D-7) to get epitopes and mimotopes of Fba protein.Methods: 1.1 Fba proteins, which were divided into four overlap peptids based on the structural domains, were truncated and expressed.The amino acid residues in 4 overlap peptides are 37~110thaa, 68~161thaa, 104~277thaa, 160~324thaa respectively. They were designated as FbaA1, FbaA2, FbaA3, FbaA4 accordingly. The products were analyzed by SDS-PAGE and purified by affinity chromatography column.1.2 Female Balb/c mice were randomly individed into 5 groups, and immunized respectively with FbaA1, FbaA2, FbaA3, FbaA4 protein, and PBS as control. Mice were immunized with the same dose at an interval of 2 weeks, and boosted twice.1.3 Blood was obtained from mice inner canthal and the sera IgG was detected by ELISA. Mice were challenged with GAS to evaluate the protective rates on day 10 after the last immunization.2.1 New Zealand rabbits were immunized four times with the purified Fba protein with the same dose at an interval of 2 weeks, and blood was obtained from carotid artery and the IgG titer was detected by ELISA. Antibodies were purified and the titer was detected by ELISA too.2.2 The antibody of GAS was used to screen the Phage display seven-peptide library. After 3 rounds of panning,individual clones were picked from LB-IpTG/xgal plates. 21 clones were randomly selected and examined by ELISA with Fba immune serum, and the positive clones were sequenced. According to sequencing, sequencing of amino acids was deduced,then compared with the sequencing of Fba protein online to determine the epitopes of Fba protein. Results: 1.1 SDS-PAGE analyses showed that the Fba1, Fba2, Fba3 and Fba4 truncated protein were expressed and purified successfully.1.2 Levels of serum IgG of each experimental group gradually increased after mice were immunized with the four truncated proteins respectively, among which the IgG titer of Fba2 protein group increased most significantly, followed by a downward Fba3, Fba4 and Fba1 protein groups.1.3 5 days after challenged, PBS group mice all died, and protective rate in each experimental group wer as follows: Fba2 protein group was 50%, Fba1, Fba3 and Fba4 protein group are all 25%, mice immunized with each truncated proteins were evoked significantly protective immune responses compared with the PBS control.2.1 Polyclonal antibodies were successfully prepared and purified.2.2 Following three rounds of panning, the high-affinity phage clones were obtained. ELISA-positive phage clones were sequenced, and according to results of sequencing, 15 liner epitopes and 3 mimotopes of Fba were deduced.Conclusions: 1. Truncated FbaA2 protien was preliminarily determined to be able to induce stronger protective immune response.2. A number of Fba protein epitopes were acquired.