Coxsackie virus A16 type virus-like particle vaccine
A coxsackie virus, A16 technology, applied in the field of biotechnology and biomedicine, can solve problems such as an ideal vaccine without coxsackie virus
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Embodiment 1
[0117] Embodiment 1, the construction of baculovirus
[0118] The gene fragments encoding P1 and 3CD of CVA16 / shzh05 were amplified and synthesized by reverse-transcribing the first-strand cDNA, and then cloned into the pFBD vector, named pFBD-CVP1 and pFBD-CV3CD, respectively. The CVP1 gene is regulated by the polyhedrin promoter in pFBD-CVP1, and the CV3CD gene is directly regulated by the p10 promoter in pFBD-CV3CD. pFBD-CVP1 / 3CD loaded with two genes at the same time ( figure 1 ) was also successfully constructed, which can co-express two proteins efficiently in the same kind of cells.
[0119] The above three structures were subsequently constructed into corresponding baculoviruses, named Bac-CVP1, Bac-3CD, and Bac-CVP1 / 3CD, respectively.
Embodiment 2
[0120] Example 2, expression of CVA16VLPs in insect cells
[0121] Sf9 insect cells were infected with Bac-CVP1, Bac-3CD and Bac-CVP1 / 3CD respectively. Firstly, the polyclonal antibody against-CVA16VPO was used to detect the expression of the target protein in infected insect cells by immunofluorescence method. The result is as figure 2 As shown in A, Bac-CVP1 and Bac-CVP1 / 3CD had bright green fluorescence; no fluorescent signal was detected in the blank group and Bac-3CD infected group. The expression of Bac-CVP1 in insect cells was also confirmed by ELISA experiments: the anti-CVA16 VP0 antibody detected the expression of Bac-CVP1 and Bac-CVP1 / 3CD cell lysis, but the blank group and Bac-3CD infection group did not detect to the signal ( figure 2 B).
[0122] Secondly, the anti-VP0 antibody Western blot analyzed the shearing process of P1 by 3CD. like figure 2 As shown in C, no bands were detected in the blank group and Bac-3CD infection group, but the lysate of Bac-...
Embodiment 3
[0125] Example 3, Immunogenicity of CVA16 virus-like particles in mice
[0126] Animal experiments were used to evaluate the immunogenicity of CVA16 virus-like particles. The crude purified virus-like particles were injected intraperitoneally at 1ug / mouse at 0, 3 and 6 weeks, with aluminum adjuvant as the adjuvant, and uninfected Sf9 cells treated in the same way as comparison. The mouse serum was collected two weeks after the last immunization, and the specific antibody was detected by recombinant antigen-coated ELISA. The results showed that the serum of the VLP immune group could react with the recombinant subunit coat protein, while the control Sf9 serum did not react ( Figure 5 ).
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