Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof
A technology of group A rotavirus and chicken egg yolk antibody, which is applied in the field of egg yolk antibody, can solve the problems of high production requirements, loose poison, and difficult purification of egg yolk antibody
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Embodiment 1
[0033] Example 1 Preparation of Rotavirus VP7 Egg Yolk Antibody and Oral Antibody Dry Powder
[0034] Include the following steps:
[0035] (1) Preparation of VP7 recombinant antigen
[0036] According to the conservation of VP7 gene sequence of human group A rotavirus, a pair of PCR amplification primers were designed: VP7 upstream primer (5'-CCGGAATTC EcoRI TTGCCAATTACTGGATCTAT-3’) and downstream primer (5’-CCGCTCGAG wxya CATTCGTTCTGTTTGTGGA-3'), using the full-length VP7 gene (obtained from the human group A rotavirus T114 strain purchased from the Institute of Microbiology, Chinese Academy of Sciences) as a template, and the annealing temperature was set at 54 °C for PCR amplification (PCR amplification program: Heat activation, 110°C; denaturation, 94°C 1min; annealing, 54°C 45s; extension, 72°C 1min). Gel electrophoresis and recover the PCR product (about 700bp), after EcoR I and Xho I Enzyme digestion recovery, and the same double digestion ( EcoR I and X...
Embodiment 2
[0046] Example 2 Preparation of Rotavirus VP8 Egg Yolk Antibody and Oral Antibody Dry Powder
[0047] Include the following steps:
[0048] (1) Preparation of VP8 recombinant antigen
[0049] According to the conservation of VP8 gene sequence of human group A rotavirus, a pair of PCR amplification primers were designed: VP8 upstream primer (5'-CCGGAATTC EcoR I ATGGCTTCACTCATTTAT-3') and downstream primer (5'-CCGCTCGAG Xho I AACTTGTGCCCTCTTATA-3 '), using the VP8 full-length gene (obtained from the human group A rotavirus T114 strain purchased from the Institute of Microbiology, Chinese Academy of Sciences) as a template, the annealing temperature was set at 56°C for PCR amplification (PCR amplification program: heat activation, 110°C; denaturation, 94°C 1min; annealing, 56°C 45s; extension, 72°C 1min). Gel electrophoresis and recovery of the PCR product (about 750bp), digested with EcoR I and Xho I, ligated with the pET28a(+) prokaryotic expression vector that was also treat...
Embodiment 3
[0059] Example 3: This example is a comparative analysis of the potency of VP7 or VP8 egg yolk antibody dry powder obtained in Examples 1 and 2 and commercially available antibodies
[0060] 1. The titer comparison between the VP7 yolk antibody prepared in Example 1 and similar antibodies
[0061]The dilution factor of the antigen VP7 was analyzed by checkerboard titration, and the results showed that the 2000-fold dilution of the VP7 antigen was the best antigen dilution factor, embedded in the microplate well, incubated overnight at 4°C, washed 3 times with PBST washing buffer, and blocked by adding 200 μL solution, let stand at 37°C for 1.5h, washed 3 times in the same way, purchased commercially available egg yolk antibody induced by group A rotavirus particles and VP7 egg yolk antibody prepared in Example 1, serially diluted, added 100 μL of corresponding antibody to each well, and incubated at room temperature for 2 ~3h; wash 3 times in the same way, add goat anti-chicke...
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