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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
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one step rt pcr kit  (Qiagen)
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template <t>for</t> <t>RT-PCR</t> to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.
Step Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template for RT-PCR to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.

Journal: Veterinary Research

Article Title: Potent neutralization and therapeutic efficacy of bovine rotavirus-specific VHH antibodies in infected calves

doi: 10.1186/s13567-026-01765-3

Figure Lengend Snippet: Construction and screening of phage display library . A PBMCs were isolated from camels, and RNA was extracted as a template for RT-PCR to amplify the gene sequence containing the leader signal sequence before the CH2 region. The amplification yielded two fragments: a 900-bp fragment (VH–CH1–CH2) and a 600-bp fragment (VHH–CH2). Lane M represents the DNA marker (2000–100 bp), and lanes 1–4 represent the amplified fragments. B The 600-bp fragment obtained from the first round of PCR was used as a template for the second round of PCR, which successfully generated the full-length VHH gene (spanning FR1 to FR4) with an approximate size of 400 bp. Lane M represents the DNA marker (1000–200 bp), and lanes 1–4 represent the amplified fragments. C A total of 16 clones were randomly selected from the constructed phage antibody library for identification. D Overall, 92 recombinant phage clones were randomly selected from the third round of panning and added to microplates coated with BRV. HRP-conjugated anti-M13 monoclonal antibody was used to detect the bound phages. M13K07 helper phage and PBS served as the negative control and blank control, respectively. Positive clones were defined as those with a sample-to-negative control ratio (P/N) ≥ 2.1, i.e., clones above the reference line were identified as positive.

Article Snippet: The extracted RNA was directly subjected to one-step reverse-transcription polymerase chain reaction (RT-PCR) amplification using the PrimeScriptTM One Step RT-PCR Kit (TaKaRa, Shiga, Japan), which allows reverse transcription and PCR amplification to be conducted in a single reaction.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Marker, Generated, Clone Assay, Construct, Recombinant, Negative Control, Control