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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

Inactive Publication Date: 2014-02-05
SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inactivated vaccines have high production requirements, incomplete inactivation and the danger of loose virus. At the same time, due to factors such as virus residues, these will bring difficulties to the purification of egg yolk antibodies in the later stage.

Method used

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  • Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof
  • Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof
  • Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a chicken egg yolk antibody resisting human A rotavirus as well as a preparation method and application thereof. The method comprises steps of expressing and purifying structural protein VP7 or VP8 of human rotavirus in vitro, immunizing laying hens, collecting eggs, and separating and purifying egg yolk antibody of VP7 or VP8. The prepared chicken egg yolk antibody can be freeze dried and prepared into antibody dry powder for preventing and treating rotavirus. The preparation method has the beneficial effects that a large quantity of VP7 and VP8 antigens are expressed and recombined in vitro by a genetic engineering technology, the benefits are high, the cost is low, small field is required, and the method is environment-friendly; the potential risk of scattering virus caused by inactivation by rotavirus or attenuated vaccine in the traditional preparation process of egg yolk antibody is avoided, and the method meets the requirement of animal welfare. The egg yolk antibody prepared by the method can be prepared into oral antibody dry powder biological agent after sterility test, has obvious effect in treating rotavirus infectious diarrhea, takes effect rapidly and has low cost, and the prevention rate reaches over 95%.

Description

technical field [0001] The invention relates to an egg yolk antibody, in particular to a high-titer chicken egg yolk antibody against group A rotavirus, a preparation method thereof and an application in preventing and treating rotavirus infection. Background technique [0002] Human group A rotavirus (Rotavirus, RV) is the main cause of infectious diarrheal diseases in infants and young children. Its incidence is high and its harm is serious, which seriously affects the development, nutritional requirements and quality of life of infants and young children [Parashar UD et al., 2003 ; Yang Yanli et al., 2011]. No matter in developed or developing countries, gastroenteritis caused by rotavirus is a serious public health problem. Diarrhea caused by rotavirus is widespread and occurs in autumn and winter. Almost all children under 5 years old suffer from An RV infection has occurred. It is estimated that there are more than 25 million outpatient cases and more than 2 million ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/02A61K39/42A61P31/14
Inventor 李再新张智胡卫东李丙超李天有邹晓阳唐文才
Owner SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING
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