9 research outputs found

    Test of QC cloning using Klenow DNA polymerase.

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    <p>(A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. (<b>C</b>) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.</p

    Test of QC cloning performed with or without heat inactivation.

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    <p>(<b>A</b>) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. (<b>B</b>) Structure of the vector and of the PCR product. (<b>C</b>, <b>D</b>) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C (<b>C</b>) or incubation at 4°C (<b>D</b>). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.</p

    Quantification of T4 DNA polymerase exonuclease activity.

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    <p><i>Sac</i>II/<i>Nde</i>I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).</p

    Test of CS length, reaction temperature and incubation time.

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    <p>(<b>A</b>) To analyze the influence of the catching sequence length on cloning efficiency, the T019 GC3F PCR product was cloned into pICH31477 (23 nucleotide CS) and pICH31480 (52 nucleotide CS). Cloning was performed using an incubation at 15°C for 0, 5, 10, 20 and 30 minutes (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence) (<b>B</b>). (<b>C</b> and <b>E</b>) Eight randomly chosen clones from reactions with the incubation times that yielded the most clones were analyzed by colony PCR using vector primers. The PCR products were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow. (<b>D</b>) To determine the optimal incubation temperature and time, the T019 GC3F PCR product was cloned into pICH31480 (52 nucleotide CS) using incubation temperature of 4°C, 15°C and 25°C for 0, 5, 10, 20, 30 and 60 minutes.</p

    Construction of QC cloning vectors.

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    <p>QC cloning vectors can be prepared by amplification of a DNA fragment containing a visible selectable marker (a <i>lacZ</i>α fragment was used here) with two primers with 5′ extensions containing the bap2 sequence (blue box) and the catching sequence (CS, red box). The primers also contain extensions C and D with homology with any cloning vector of choice. The PCR product is cloned by ligation-independent cloning in a linearized vector (here pICH36833) with DNA ends homologous to sequences C and D. DNA from blue colonies are sequenced to make sure that no mutations are present in the sequence of bap2 and the CS.</p

    Cloning of Gamma, Mu, Kappa and Lambda immunoglobulin fragments by blunt-end and QC cloning.

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    <p>(<b>A</b>) PCR products amplified from G-tailed cDNA prepared from non-Hodgkin lymphoma biopsy sample T019 (isotype Gamma, Lambda) and T069 (isotype Mu, Kappa). Amplification was performed using primer bap2 pc and primers GC3F, LC1N, Mu1F and KC2F as indicated. The PCR products were cloned using QC cloning (<b>B and D</b>) or blunt-end cloning (<b>C and E</b>). 12 randomly chosen clones for each reaction were analyzed by colony PCR using vector primers. The expected size of full-length inserts is indicated by a dashed line and an arrow.</p

    Principle of the QC cloning strategy.

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    <p>(<b>A</b>) PCR products amplified to identify unknown sequences flanking a region of known sequence typically consist of an adaptor sequence (A) attached to the end of the unknown sequence (U) followed by a region of known sequence (K). The PCR product is amplified with two primers (1 and 2) that are homologous to the adaptor sequence and to part of the known sequence (region K2). Non-specific products (ns) and primer dimers can also be obtained during PCR amplification. (<b>B</b>) The fragment is cloned by homology with a linearized vector that is homologous to the adaptor sequence at one end and to a sequence from the known region (K1, called the CS in the cloning vector) at the other end. Since sequence K1 does not overlap with sequence K2, non-specific products and primer dimers cannot be cloned.</p

    Comparison of blunt-end cloning, ligation-independent cloning and QC cloning on sample T044.

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    <p>(<b>A</b>) To compare the efficiency of the three cloning methods, a PCR product amplified from G-tailed cDNA prepared from sample T044 was cloned using blunt-end, LIC and QC cloning (A, adaptor; U, unknown sequence; K, known sequence; ns, non-specific; CS, catching sequence). (<b>B</b>) PCR product amplified from T044 G-tailed cDNA using primers bap2 pc and cga3.1. (<b>C, D, E</b>) 12 randomly picked clones for each cloning strategy were analyzed by colony PCR using vector primers. The size of the expected immunoglobulin fragment is indicated by an arrow and a dashed line.</p

    DataSheet_1_A randomized, double-blind, placebo-controlled, dose-escalating phase I trial to evaluate safety and immunogenicity of a plant-produced, bivalent, recombinant norovirus-like particle vaccine.docx

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    Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide and a safe and effective vaccine is needed. Here, a phase I, double-blind, placebo-controlled clinical trial was performed in 60 healthy adults, 18 to 40 years old. Safety (primary objective) and immunogenicity (secondary and exploratory objectives) of a bivalent (GI.4 and GII.4), plant-produced, virus-like particle (VLP), NoV vaccine candidate formulation were investigated at two dose levels (50 µg + 50 µg and 150 µg + 150 µg) without adjuvant. Overall, 13 subjects (65.0%) in the 50 µg group, 16 subjects (80.0%) in the 150 µg group, and 14 subjects (70.0%) in the placebo group reported at least 1 solicited local or general symptom during the 7-day post-vaccination periods following each dose. Severe solicited adverse events (AEs) were rare (2 events in the 50 µg group). A total of 8 subjects (40.0%) in each group reported at least one unsolicited AE during the 28-day post-vaccination periods. Immunogenicity was assessed on days 1, 8, 29, 57, 183 and 365. All subjects were pre-exposed to norovirus as indicated by baseline levels of the different immunological parameters examined. Vaccine-specific humoral and cellular immune responses increased after the first dose but did not rise further after the second vaccination. Increased GI.4- and GII.4-specific IgG titers persisted until day 365. The vaccine elicited cross-reactive IgG antibodies against non-vaccine NoV VLPs, which was more pronounced for NoV strains of the same genotype as the GII.4 vaccine strain than for non-vaccine genotypes. Significant blocking anti-GI.4 and anti-GII.4 VLP titers were triggered in both dose groups. Lymphoproliferation assays revealed strong cell-mediated immune responses that persisted until day 365. In conclusion, both dose levels were safe and well-tolerated, and no higher incidence of AEs was observed in the higher dose group. The data show that a single dose of the vaccine formulated at 50 µg of each VLP is sufficient to reach a peak immune response after 8 to 28 days. The results of this Phase I study warrant further evaluation of the non-adjuvanted vaccine candidate.Clinical trial registrationhttps://clinicaltrials.gov/ct2/show/record/NCT05508178, identifier (NCT05508178).</p
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