Preparation and Application of Coxsackievirus a6 Virus-Like Particles in Insect Cells
A virus-like, virus-based technology, applied in the field of biomedicine, can solve problems such as the lack of vaccines for hand, foot and mouth disease
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0111] 1.6 Preparation and purification of VLP
[0112] After the successful expression was verified by a small amount of detection, the P3 generation baculovirus was used for mass expression of VLP. First, insect cells Sf9 (Invitrogen) were infected with P3 generation baculovirus, harvested after 72 hours and centrifuged to separate the medium from the cells, and the precipitated cells were lysed and broken with Tris-Nacl buffer containing 1% NP-40 to obtain the lysed The solution was concentrated by ultracentrifugation on a 20% sucrose pad at 27,000 rpm for 6 hours, and the resulting precipitate was resuspended with Tris-Nacl. Then, through 10%-50% sucrose density gradient centrifugation, the condition is 39,000 rpm, 2.5h, after sucrose density gradient centrifugation, 12 fractions of equal volume are taken from top to bottom to analyze the VLP content. The VLP-rich fractions were collected and mixed, and then concentrated by ultracentrifugation on a 20% sucrose pad, the co...
Embodiment 1
[0133] Expression of embodiment 1CA6-VLP
[0134] In order to be able to co-express the P1 and 3CD proteins of CA6, the inventors inserted the P1 and 3CD coding polynucleotide sequences together into the pFBD vector, and the P1 and 3CD were respectively located behind the pPH and pP10 promoters to obtain pFBD-CA6-P1 / 3CD , whose structure is shown in the figure 1 As shown in A. This plasmid was then used to transform DH10Bac TM Competent cells obtain Bac-CA6-P1 / 3CD for subsequent expression. The present inventor adopts Bac-to-Bac insect baculovirus expression system, transfects Sf9 cell with Bac-CA6-P1 / 3CD, uses the Sf9 cell of untransfected plasmid as control, uses indirect ELISA ( figure 1 B) and Western Blot ( figure 1 C) detection of the expression of CA6-P1 / 3CD, anti-CA6-VP0, anti-CA6-VP1 and anti-CA6-VP3 three kinds of antibody detection all show that the cell lysate of transfection Bac-CA6-P1 / 3CD can produce very strong Specific reaction, while the Sf9 cells not tra...
Embodiment 2
[0135] Assembly and antigenic identification of embodiment 2CA6-VLP
[0136] In order to identify the expression and assembly of CA6-VLP, Bac-CA6-P1 / 3CD transfected Sf9 and blank Sf9 cell lysates were subjected to 10%-50% sucrose density gradient centrifugation after being passed through a 20% sucrose pad, from top to bottom Take 12 layers, and use indirect ELISA and Western Blot to detect the protein content of each layer. The results of ELISA analysis showed that there were more VPO, VP1 and VP3 proteins in layers #6, #7 and #8 ( figure 2 A). The results of Western Blot detection also showed that anti-CA6-VP0, VP1 and VP3 were used as antibodies, and strong specific reactions could be produced in these three layers ( figure 2 B). The layers rich in VLP were collected and mixed, and then concentrated as a 20% sucrose cushion to obtain a CA6-VLP vaccine with better purity and higher concentration. Get this solution and analyze with transmission electron microscope, can o...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More