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Preparation method and application of specific antibody containing milk or serum produced by novel coronavirus immunized cows

A coronavirus and immune preparation technology, applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problems of not being popularized, not suitable for blood donation, restrictions, etc., to reduce mortality, prevent virus from invading cells, The effect of reducing virus content

Inactive Publication Date: 2020-11-20
HUAZHONG AGRI UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical use of serum from recovered patients with novel coronavirus pneumonia has a certain curative effect on critically ill patients, but the method of using plasma from recovered patients to treat viral infections has not been popularized. On the other hand, it is also limited by factors such as plasma sources

Method used

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  • Preparation method and application of specific antibody containing milk or serum produced by novel coronavirus immunized cows
  • Preparation method and application of specific antibody containing milk or serum produced by novel coronavirus immunized cows
  • Preparation method and application of specific antibody containing milk or serum produced by novel coronavirus immunized cows

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Preparation of milk and serum containing novel coronavirus antibodies:

[0028] The sequence of the novel coronavirus hCov-19 / Wuhan / ZY38-1 / 2020 strain was submitted to the GISAID database on July 10, 2020, and the accession number is EPI_ISL_486645. In the present invention, the virus is referred to as novel coronavirus ZY38-1 for short.

[0029] 1. Proliferation, culture and inactivation of novel coronavirus ZY38-1

[0030] (1) Cell culture:

[0031] VERO cells were cultured in DMEM containing 10% FBS for about 48 hours;

[0032] Cell subculture: digest with 0.25% trypsin during subculture, and the cells can be digested in about 10 minutes.

[0033] Specific steps are as follows:

[0034] For adherent cells, use a dropper or pipette to suck off the old culture medium in the culture vessel, and wash away the residual old culture medium with serum-free DMEM medium;

[0035] Add 1-2ml of digestive solution (0.25% trypsin) to the bottle and gently shake the culture bot...

Embodiment 2

[0056] Determination of Specific Antibody Levels in Immune Milk and Immune Serum

[0057] 1. Expression and purification of novel coronavirus RBD antigen protein

[0058] Through the design of expression elements and vector reconstruction, the RBD gene of the new coronavirus was inserted into the pCMV3 expression vector to construct the pCMV3-RBD-his eukaryotic expression vector, and the target protein was obtained by transient expression in 293T suspension cells. 1.1 Construction of pCMV3-RBD-his plasmid

[0059] Extract inactivated ZY38-1 virus RNA, reverse transcription and amplify viral cDNA, use cDNA template for RBD fragment amplification, amplification primer RBD-F: 5'-ATGAGGGTCCAACCAACAGAGAGC-3'; RBD-R: 5'-GAAGTTCACACACTTGTTCTTCAC -3'. In order to facilitate subsequent RBD protein purification, 6×HIS sequence was added to the back end of RBD gene to obtain RBD-his gene. The pCMV3 empty vector and RBD-his gene were double digested with KpnI and XbaI, and the fragment...

Embodiment 3

[0083] Neutralizing antibody titer of immune milk and immune serum

[0084] The new coronavirus plaque reduction micro-neutralization test was used to detect the neutralizing antibody titer of the antibody in the immune milk and immune serum to the new coronavirus. The specific steps are as follows:

[0085] (1) Passage Vero cells in good growth state to 12-well cell culture plate, 3-5E5 / well, and grow into a dense monolayer the next day.

[0086] (2) Inactivate the serum and milk samples processed in Example 2 and stored at -20°C for 30 minutes at 56°C.

[0087] (3) Serum and milk samples were diluted with serum-free DMEM medium in a 24-well plate, and the number of 24-well plates was prepared according to the number of samples, with 4 samples per plate, and a virus back-titration control was added.

[0088] (4) Add 441ul or 405ul DMEM to the first column of each well, and add 300ul to other wells.

[0089] (5) Add 9 ul of serum or 45 ul of milk sample to the wells in the f...

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Abstract

The invention belongs to the field of biotechnology, and discloses a preparation method and application of specific antibody containing milk or serum produced by novel coronavirus immunized cows. Theinactivated novel coronavirus hCov-19 / Wuhan / ZY38-1 / 2020 is used for immunizing dairy cows, and a large amount of immune milk and serum can be obtained. Both the immune milk and the serum contain highcontent of antibodies, viruses can be effectively neutralized and stopped from invading cells, and the significant anti-SARS-CoV-2 virus effect is achieved; after Igs protein purified from immune milkis injected into mice, the mortality of novel coronavirus infected mice can be significantly reduced, the influence of novel coronavirus infection on weight loss of the mice is reduced, and the content of viruses in lung tissue of the mice is reduced. The immunized milk and serum can be used as sources of antibodies for preparations for preventing and treating novel coronavirus pneumonia, and thelimitation of the sources of plasma for rehabilitation of novel coronavirus pneumonia.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of specific antibody-positive milk and serum for severe acute respiratory syndrome coronavirus 2. Background technique [0002] The novel coronavirus pneumonia (coronavirus disease 19, COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) appeared at the end of 2019 and presented a global outbreak, seriously endangering human beings Health and public health security have brought huge burdens to the world society and economy, and have aroused great concern worldwide. As of the end of July 2020, there have been more than 14 million confirmed cases of new coronary pneumonia worldwide, and more than 600,000 deaths. Many domestic and foreign scholars speculate that the new coronavirus may be similar to influenza virus and coexist with humans for a long time. Moreover, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K16/04C07K16/06A01K67/02A61K39/42A61K39/395A61P31/14
CPCC07K16/10C07K16/04C07K16/06A01K67/02A61P31/14A61K2039/505
Inventor 金梅林杨丽黄坤回显峰陈洁陈焕春涂亚平康超刘武张池孙小美邹忠万平民赵亚陈西吴超金尔光
Owner HUAZHONG AGRI UNIV
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