[Development of a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri].

OBJECTIVE: To develop a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella (S.) flexneri. METHODS: Eight pairs of primer for O-antigen synthesis and modification genes of S. flexneri were designed and used for developing an O-antigen modification gene-specific singleplex PCR assay to serotype 14 most common S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Y, X, Xv and F6). Bacterial pathogens which causing diarrheal disease were used for specificity detection. 106 S. flexneri clinical isolates were serotyped by this method and compared with the slide agglutination method. RESULTS: An O-antigen modification, gene-specific singleplex PCR was developed. When six singleplex PCR reactions were performed, 14 of the 15 recognized S. flexneri serotypes were identified, except for serotype Xv. The detection threshold ranged from 10 pg to 1 ng DNA in a 20 µl reaction system. A high concordance between the singleplex PCR assay and slide agglutination were observed when 106 S. flexneri strains of various serotypes were analyzed with an exception that 1 serotype Y strain showed that it was carrying the additional defective gtr II genes. CONCLUSION: This method showed advantages over the traditional slide agglutination methods, and was promising when under application in the following situations as clinical diagnosis.

[Identification of Haemaphysalis longicornis and Rhipicephalus microplus].

OBJECTIVE: To establish a method combined morphology and molecular marker for identifying Haemaphysalis longicornis and Rhipicephalus microplus. METHODS: Ticks were collected from domestic animals and wild environment in epidemic area of Hubei and Henan provinces where cases of fever with thrombocytopenia syndrome were prevalent. We classified the ticks by morphology characteristics before 12S rDNA of ticks were amplified by PCR and subsequently sequenced. Phylogenetic tree was constructed by PAUP4.0. RESULTS: The ticks belonged to Haemaphysalis longicornis and Rhipicephalus microplus through observation and analysed by the morphological characteristics of the ticks. 12S rDNA was cloned and sequenced while data confirmed the morphological identification of the results. CONCLUSION: The method based on morphology that combined with molecular marker seemed a good method for the identification of ticks.

[Study on the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains].

OBJECTIVE: To study the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains. METHODS: A series of primers were designed based on potential integration site of SfII and SfX prophages in Shigella flexneri serotype 2b strains, and PCR were performed for 50 serotype 2b strains to amplify special genes located in host and prophages. PCR products were sequenced to identify integration sites and arrangement of SfII and SfX. RESULTS: In all the serotype 2b strains, prophage SfII and SfX were adjacent to each other, and integrated into the thrW tRNA gene of the host, which were located between genes proA and yaiC of host. Prophage SfX was located immediately upstream of prophage SfII in all the detected 50 serotype 2b strains exception for strain 51251. CONCLUSION: This was the first report on the integration site and arrangement of serotype-converting prophages SfII and SfX in Shigella flexneri 2b strains.

[Secretion expression of cholera toxin B subunit in food-grading Lactococcus lactis expression system].

OBJECTIVE: To clone and secretion express cholera toxin B subunit (CTB) in food-grading Lactococcus lactis expression systems. METHODS: ctB fragment that encoding CTB was amplified by polymerase chain reaction (PCR) using the genomic DNA of Vibrio cholera strain 569B as template and was inserted into two secretion expression vector pSQZ and pSQ to construct food-grading expression system L.lactis MBP71/pSQZ-ctB and L.lactis MBP71/pSQ-ctB. The expressed CTB was detected by Western-blot assay. RESULTS: The ctB fragment was successfully amplified from Vibrio cholera strain 569B and inserted into two secretion expression vectors pSQZ and pSQ to construct food-grading expression system L. lactis MBP71/pSQZ-ctB and L. lactis MBP71/pSQ-ctB. Western-blot assay demonstrated that CTB was secretion and expressed from L.lactis MBP71 harboring vectors pSQZ-ctB and pSQ-ctB, and the quantity of CTB secreted by L. lactis MBP71/pSQ-ctB was about 2 microg/ml, higher than that of L. lactis MBP71/pSQZ-ctB. CONCLUSION: CTB was successfully secreted and expressed by food-grading L. lactis expression systems.

[Clone and express ctb-stx2b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeal toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity].

OBJECTIVE: To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157 : H7 (EHEC O157 : H7) Shigela toxin 2B subunit (Stx2B) and vibrio cholera toxin B subunit (CTB) as well as to detect the immunogenicity and GM1-binding ability of fusion protein. METHODS: To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified, then to transform constructed plasmid into E. coli BL21 (DE3) induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDS-PAGE and Western-blot. RESULTS: The amplified ctb-stx2b fragments appeared to be 750 bp and gene sequence was identical to designed sequence. The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about M(r) 20 x 10(3) and the expressed protein could react to CTB monoclone anti-body. The fusion protein CTB-Stx2B could bind GM1. CONCLUSION: CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability. This study provided information on further EHEC O157 : H7 vaccine research.

[Immunoproteomic assay of secretive proteins from Streptococcus suis type 2 strain SC84].

OBJECTIVE: To identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84. METHODS: Two-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005. RESULTS: A total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005. CONCLUSION: The newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.