Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Plasmid Preparation, Clone Assay, Expressing, TA Cloning, Sequencing, Derivative Assay

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Diagram of H. ducreyi hemolysin gene region from strain 35000, the hemolysin-negative derivative of strain 35000, constructs containing the hhdA and hhdB genes, and locations of primers and probes used in this study. Restriction sites used for cloning that were added as a result of PCR amplification or from multiple cloning sites are underlined. The cat cassette, used to replace the PstI fragment internal to the hhdA gene in 35000ΔAPC, is necessarily not drawn to scale. U32175 is the GenBank accession number for hhdB and hhdA (35); numbers on the diagram indicate base pairs.

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Construct, Clone Assay, Amplification

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Identification of H. ducreyi isolates, detection of homology to hemolysin genes, and phenotypic expression of hemolysin in geographically and temporally diverse isolates of H. ducreyi. (A to C) Spot blots of H. ducreyi and other Haemophilus species probed with whole-cell DNA from H. ducreyi type strain CIP 542 (A), the hhdB gene (B), and the hhdA gene (C). Strains and species analyzed: top row, CIP542, 35000, LA228R, CF101, V-1168, V149/91, V-180, HMC56, CHIA, and V-1157; bottom row, H. influenzae Rd, H. parainfluenzae, H. aphrophilus, H. paraphrophilus, H. segmis, A. pleuropneumoniae, H. haemoglobinophiles, T. equigenitalis, H. haemolyticus, and H. influenzae ATCC 33911. (D) Hemolysis of H. ducreyi isolates on HBAPs. Isolates on HBAPs correspond to those in the top rows of panels A to C except strain 35000-KmA, the right-most strain in panel D, which was added as a nonhemolytic control. The photographs of the Southern blots and the HBAP were scanned with a Microtek Scanner III and captured with a Micrografx Picture Publisher (Micrografx, Richardson, Tex.).

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Expressing

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Purification of His-tagged hemolysin from strain 35000-KmA(pLSBAHis+) demonstrated by SDS-PAGE analysis (A) and Western blot analysis with rabbit polyclonal serum against His-tagged hemolysin (B). Lanes: 1, strain 35000-KmA(pLSBAHis+) cell lysate; 2, purified hemolysin from strain 35000-KmA(pLSBAHis+) eluted from Ni-NTA resin; 3, strain 35000-KmA(pLSSK) cell lysate; lane 4, strain 35000-KmA(pLSSK) cell lysate eluted from Ni-NTA resin. Twenty-five micrograms of each cell lysate and equal volumes of Ni column eluates were analyzed on the gel and Western blot. Positions of the H. ducreyi hemolysin protein (arrows) and of the molecular weight protein standards are indicated. Both images were photographed with a Bio-Rad GelDoc 2000 digital camera system equipped with Quantity One 4.0 software (Bio-Rad).

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Purification, SDS Page, Western Blot, Molecular Weight, Software

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Production of antibodies to hemolysin among rabbits and monkeys infected with H. ducreyi. Each lane contains 200 ng of purified His-tagged hemolysin separated on an 8% polyacrylamide gel, blotted to nitrocellulose, and probed with pooled preimmune rabbit serum (lane 1), serum from rabbits infected three times over 3 months with H. ducreyi 35000 (lane 2), preimmune monkey serum (lane 3), and serum obtained from the same monkey 6 weeks after inoculation of strain CH2 (lane 4).

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Infection, Purification

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Lesion scores for control and immunized rabbits infected with H. ducreyi 35000 and 35000ΔAPC

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: Infection

Journal:

Article Title: Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

doi:

Figure Lengend Snippet: Immunization with hemolysin decreases recovery of H. ducreyi 35000 but not 35000ΔAPC in the temperature-dependent rabbit model

Article Snippet: E. coli BL21(DE3) Host for protein expression Novagen, Madison, Wis. H. aphrophilus ATCC 33389 Arnold Smith H. ducreyi 35000-TcA Strain 35000 with Tn 916 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000-KmA Strain 35000 with Tn 1545-Δ3 inserted in hhdB , nonhemolytic 50 H. ducreyi 35000ΔAPC Strain 35000 with cat cassette in hhdA gene, nonhemolytic 54 H. haemoglobinophilus ATCC 19416 ATCC, Manassas, Va. H. haemolyticus ATCC 33390 Arnold Smith H. influenzae ATCC 33911 Arnold Smith H. influenzae Rd Marilyn Roberts H. parainfluenzae ATCC 33392 Arnold Smith H. paraphrophilus ATCC 29237 Arnold Smith H. segmis ATCC 33393 Arnold Smith T. equigenitalis ATCC 35865 Arnold Smith Plasmids pUC19 E. coli cloning vector, Ap r GibcoBRL pCR2.1 TA cloning vector for cloning PCR products, Amp r Kan r Invitrogen, San Diego, Calif. pET24+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, no translation initiation signals, Kan r Novagen pET24a+ Expression plasmid with C-terminal His tag sequence and inducible T7 promoter, Kan r Novagen pBCKS Cm r plasmid derived from pUC19 with KS multiple cloning site Stratagene, La Jolla, Calif. pPT384-ETa hhdBA genes cloned into pET24a+ Sst I- Sal I This study pLSSK Shuttle vector with ori , sulA , and strA genes from pLS88 and multiple cloning sites and lacZ gene from pBluescript SK 54 pPT384 hhdBA gene region in pTZ18 in same orientation as lac promoter 50 pPT376BCKS 5.8-kb Bgl II fragment containing part of hhdB and all of hhdA cloned into pBCKS Sst I- Sal I 50 pETBABN hhdBA genes cloned into pET24+ Bam HI- Sal I This study pLSBAHis+ hhdBA genes cloned into pLSSK in same orientation as lac promoter.

Techniques: