Bvh-a2 and bvh-a3 antigens of group b streptococcus
a technology of streptococcus and antigens, which is applied in the field of polypeptides of group b streptococcus, can solve the problems of postpartum infection, death, disability, and recurrence of infection of expectant mothers exposed to gbs
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example 1
[0153]This example illustrates the identification of GBS BVH-A2 and BVH-A3 genes.
[0154]Chromosomal DNA was isolated from different GBS strains as previously described (Jayarao B M et al. 1991. J. Clin. Microbiol.29: 2774-2778). A λZAPExpress genomic library was constructed using chromosomal DNA purified from the serotype III GBS strain NCS954 and screened according to the manufacturer's instruction (STRATAGENE, La Jolla, Calif.) with a pool of human normal sera. Briefly, the purified chromosomal DNA was partially digested with tsp509I restriction enzyme, and the resulting fragments were electrophoresed on a 1% agarose gel (Bio-Rad). Fragments in the 5-to 10-kb size range were extracted from the gel and ligated to the EcoRI arms of λZAPExpress vector and the vector was encapsidated using the Gigapack II packaging extract (STRATAGENE). The recombinant phages were used to infect E. coli XLI-Blue MRF′ [Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-1 recA1 gyrA96 relAl lac (F′ proAB ...
example 2
[0155]This example illustrates the cloning of GBS BVH-A2 and BVH-A3 genes.
[0156]The coding regions of Group B streptococcal BVH-A2 (SEQ ID NO: 1) and BVH-A3 (SEQ ID NO: 5) genes were respectively amplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, Calif.) from purified recombinant phagemid clone H31-29 and genomic DNA of serotype III Group B streptococcal strain NCS954 using oligonucleotide primers that contained base extensions for the addition of restriction sites Ndel (CATATG) and Xhol (CTCGAG). The oligonucleotide primers (Table 1) DMAR172 (SEQ ID NO:9) and DMAR173 (SEQ ID NO:10) were used to amplify the BVH-A2 gene, while DMAR204 (SEQ ID NO:15) and DMAR205 (SEQ ID NO:16) were used to amplify the BVH-A3 gene. PCR products were purified from agarose gel using a QIAQUICK gel extraction kit from QIAGEN following the manufacturer's instructions (Chatsworth, Calif.), and digested with Ndel and Xhol (Pharmacia Canada Inc, Baie d'Urfé, Canada). The pET-...
example 3
[0159]This example describes the PCR amplification of GBS BVH-A2 and BVH-A3 genes from other GBS strains
[0160]To confirm the presence by PCR amplification of BVH-A2 (SEQ ID NO:1) and BVH-A3 (SEQ ID NO:5) genes, the following 11 serologically distinct GBS strains were used: C388 / 90 (serotype Ia / c), ATCC12401 (serotype Ib), ATCC27591 (serotype Ic), NCS246 (serotype II / R), NCS954 (serotype III), NCS97SR331 (serotype IV), NCS535 (serotype V), NCS9842 (serotype VI), NCS7271 (serotype VII), NCS970886 (serotype VIII), ATCC27956 (bovine isolate). These strains were obtained from the American Type Culture Collection (Rockville, Md., USA) and National Centre for Streptococcus, Provincial Laboratory of Public Health for Northern Alberta (Edmonton, Canada). The E. coli strain XL1-Blue MRF′ was used in these experiments as negative control. Chromosomal DNA was isolated from each Group B streptococcal strain as previously described (Jayarao B M et al. 1991. J. Clin. Microbiol. 29:2774-2778). BVH-...
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