Journal: Virology Journal

Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

doi: 10.1186/s12985-020-01388-w

Figure Lengend Snippet: Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, and identification of the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT. A, Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, a coding sequence (CTLT) of T lymphocyte epitopes from H1HA, H9HA, and H7HA AIV subtypes was controlled by the baculovirus P10 promoter, and a coding sequence (THB) of B cell epitopes from the H1HA, H9HA, and H7HA AIV subtypes was driven by the CMV promoter. B-1, control BmN cells; B-2, the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT-infected BmN cells. C-1, the CMV promoter amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lane 1: BmNPV; and lanes 2–4: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT. C-2, the THB fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV. C-3, the CTLT fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV

Article Snippet: CTLT (Figure S ) and THB (Figure S ) codon-optimized coding sequences based on the preference of the BmNPV codons were synthesized by GenScript Biotech Corp (Nanjiang, China), and cloned into the vector, pUC57-T (GenScript, Nanjiang, China), to generate pUC57-CTLT and pUC57-THB, respectively.

Techniques: Plasmid Preparation, Recombinant, Sequencing, Control, Infection, Amplification, Marker

Journal: Virology Journal

Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

doi: 10.1186/s12985-020-01388-w

Figure Lengend Snippet: CTLT expressed in BmNPV-CMV/THB-P10/CTLT-infected BmN cells. a CTLT transcript detected by RT-PCR; Lane M: DNA marker; Lane 1: CTLT transcript. b CTLT expressed in the BmNPV-CMV/THB-P10/CTLT-infected BmN cells was detected by Western blot; lane 1: BmN cells infected with BmNPV-CMV/THB-P10/CTLT; lane 2: BmN cells infected with BmNPV; lane 3: normal BmN cells. Primary antibody; lane M: protein marker. Mouse anti-H7H9 HA was used as a primary antibody; HRP-conjugated goat anti-mouse IgG was used as a secondary antibody. c , CTLT expressed in BmNPV-CMV/THB-P10/CTLT-infected BmN cells was detected by immunohistochemistry. 1, 1′ and 1″, normal BmN cells; 2, 2’and 2″, BmNPV-CMV/THB-P10/CTLT infected BmN cells; 1 and 2, the cell nucleus was stained by DAPI; 1′ and 2′, CTLT stained by Cy3; 1″, the merger of 1 and 1′; 2″, the merger of 2 and 2′. Mouse anti-H7H9 HA was used as a primary antibody; Cy3-labeled goat anti-mouse IgG was used as a secondary antibody

Article Snippet: CTLT (Figure S ) and THB (Figure S ) codon-optimized coding sequences based on the preference of the BmNPV codons were synthesized by GenScript Biotech Corp (Nanjiang, China), and cloned into the vector, pUC57-T (GenScript, Nanjiang, China), to generate pUC57-CTLT and pUC57-THB, respectively.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Marker, Western Blot, Immunohistochemistry, Staining, Labeling

Journal: Virology Journal

Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

doi: 10.1186/s12985-020-01388-w

Figure Lengend Snippet: THB expressed in the splenocytes of BmNPV-CMV/THB-P10/CTLT-injected chickens. The spleens were dissected from the chickens at 0, 6, 24, 48, and 72 h post-injection with BmNPV-CMV/THB-P10/CTLT, and immunohistochemistry was carried out with the mouse anti-H7H9 HA (primary antibody) and FITC-labeled goat anti-mouse IgG (secondary antibody). The cell nucleus was stained with DAPI

Article Snippet: CTLT (Figure S ) and THB (Figure S ) codon-optimized coding sequences based on the preference of the BmNPV codons were synthesized by GenScript Biotech Corp (Nanjiang, China), and cloned into the vector, pUC57-T (GenScript, Nanjiang, China), to generate pUC57-CTLT and pUC57-THB, respectively.

Techniques: Injection, Immunohistochemistry, Labeling, Staining

Journal: Virology Journal

Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

doi: 10.1186/s12985-020-01388-w

Figure Lengend Snippet: A specific immune response against THB could be generated in the chickens vaccinated with BmNPV-CMV/THB-P10/CTLT. a The detected levels of anti-THB antibodies in the serum of the vaccinated chickens with BmNPV-CMV-THB-CTLT. The chickens were vaccinated with BmNPV-CMV-THB-CTLT at a dose of 10 12 TCID 50 /kg (Group 1), 5 × 10 11 TCID 50 /kg (Group 2), and 10 11 TCID 50 /kg (Group 3). The serum samples at 1, 2, 3, 4, and 5 weeks post-vaccination were used to determine the level of anti-THB antibodies via an indirect ELISA. b The splenic T lymphocyte proliferative responses in vaccinated chickens. The lymphocyte cell suspension was stimulated with ConA (5 μg/mL final concentration) and H7HA standard antigen (0.5 μg/mL final concentration) was seeded into a 96-well plate at 100 μL per well. An unstimulated cellular suspension was used as a control. The cells were incubated at 5% CO 2 and 39.5 °C for 44 h. The cell number was determined using the MTT method. * P < 0.05

Article Snippet: CTLT (Figure S ) and THB (Figure S ) codon-optimized coding sequences based on the preference of the BmNPV codons were synthesized by GenScript Biotech Corp (Nanjiang, China), and cloned into the vector, pUC57-T (GenScript, Nanjiang, China), to generate pUC57-CTLT and pUC57-THB, respectively.

Techniques: Generated, Indirect ELISA, Suspension, Concentration Assay, Control, Incubation

Journal: Virology Journal

Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

doi: 10.1186/s12985-020-01388-w

Figure Lengend Snippet: THB expressed in the splenocytes of the BmNPV-CMV/THB-P10/CTLT-injected mice and the level of anti-THB antibody detection. a THB expressed in mouse splenocytes. The spleens were dissected from the mice at 48 h post-vaccination with BmNPV-CMV/THB-P10/CTLT, and immunohistochemistry was performed with the mouse anti-H7H9 HA (primary antibody) and FITC-labeled goat anti-mouse IgG (secondary antibody). The cell nucleus was stained with DAPI. CK, normal mouse; Test, the mice vaccinated with BmNPV-CMV/THB-P10/CTLT. b The level of anti-THB antibody detected in the serum of the vaccinated mice with BmNPV-CMV-THB-CTLT. The mice were vaccinated with BmNPV-CMV-THB-CTLT at a dose of 10 10 TCID 50 /kg (Group 1), 5 × 10 9 TCID 50 /kg (Group 2), and 10 9 TCID 50 /kg (Group 3). The serum sampled at 1, 2, 3, and 4 weeks post-vaccination were used to determine the level of anti-THB antibodies by indirect ELISA

Article Snippet: CTLT (Figure S ) and THB (Figure S ) codon-optimized coding sequences based on the preference of the BmNPV codons were synthesized by GenScript Biotech Corp (Nanjiang, China), and cloned into the vector, pUC57-T (GenScript, Nanjiang, China), to generate pUC57-CTLT and pUC57-THB, respectively.

Techniques: Injection, Immunohistochemistry, Labeling, Staining, Indirect ELISA