Novel coronavirus, preparation method for specific-antibody-containing milk or serum produced by dairy cow immunized by novel coronavirus and application of specific-antibody-containing milk or serum

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 the impact, prevent the virus from invading cells, and reduce the Mortality Effect

Active Publication Date: 2021-09-24
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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  • Novel coronavirus, preparation method for specific-antibody-containing milk or serum produced by dairy cow immunized by novel coronavirus and application of specific-antibody-containing milk or serum
  • Novel coronavirus, preparation method for specific-antibody-containing milk or serum produced by dairy cow immunized by novel coronavirus and application of specific-antibody-containing milk or serum
  • Novel coronavirus, preparation method for specific-antibody-containing milk or serum produced by dairy cow immunized by novel coronavirus and application of specific-antibody-containing milk or serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Preparation of milk and serum containing novel coronavirus antibodies:

[0030] The sequence of the novel coronavirus strain was submitted to the GISAID database on July 10, 2020, the accession number is EPI_ISL_486645, and the preservation number in the China Type Culture Collection Center is CCTCC NO:V202107; in the present invention, the virus is referred to as the novel coronavirus ZY38-1.

[0031] 1. Proliferation, culture and inactivation of novel coronavirus ZY38-1

[0032] (1) Cell culture:

[0033] VERO cells were cultured in DMEM containing 10% FBS for about 48 hours;

[0034] Cell subculture: digest with 0.25% trypsin during subculture, and the cells can be digested in about 10 minutes.

[0035] Specific steps are as follows:

[0036] 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;

[0037] Add 1-2mL of digestive solution (0.2...

Embodiment 2

[0059] Determination of Specific Antibody Levels in Immune Milk and Immune Serum

[0060] 1. Expression and purification of novel coronavirus RBD antigen protein

[0061] 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

[0062] 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 purification of RBD protein, 6×HIS sequence was added to the rear 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 fragm...

Embodiment 3

[0086] Neutralizing antibody titer of immune milk and immune serum

[0087] The neutralizing antibody titer of the antibodies in the immune milk and immune serum to the new coronavirus was detected by the new coronavirus plaque reduction micro-neutralization experiment. The specific steps are as follows:

[0088] (1) Passage Vero cells in good growth state to a 12-well cell culture plate, 3-5E5 / well, and grow into a dense monolayer the next day.

[0089] (2) Inactivate the serum and milk samples processed in Example 2 and stored at -20°C for 30 minutes at 56°C.

[0090](3) Serum and milk samples were diluted in a 24-well plate with serum-free DMEM medium, 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.

[0091] (4) Add 441 μL or 405 μL DMEM to the first column of each well plate, and add 300 μL to other wells.

[0092] (5) Add 9 μL of serum or 45 μL of milk sample to the...

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Abstract

The invention belongs to the technical field of biology and discloses a novel coronavirus, a preparation method for specific-antibody-containing milk or serum produced by a dairy cow immunized by the novel coronavirus and application of the specific-antibody-containing milk or serum. A large volume of immunized milk and serum can be obtained through immunizing the dairy cow by using an inactivated novel coronavirus SARS-CoV-2 / ZY38-1, both the immunized milk and the serum contain high-content antibodies, viruses can be effectively neutralized, the viruses are prevented from invading cells, and the immunized milk and serum have an obvious SARS-CoV-2 virus resisting effect; and after an Igs protein purified from the immunized milk is injected into a mouse, the death rate of the mouse infected by the new coronavirus can be remarkably reduced, the influence of new coronavirus infection on the weight loss of the mouse is reduced, and the content of viruses in lung tissue of the mouse is lowered. The immunized milk and serum provided by the invention can be used as an antibody source of preparations for preventing and treating novel-coronavirus pneumonia, and the limitation of sources of rehabilitation blood plasma for the novel-coronavirus pneumonia is made up.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of milk and serum positive for severe acute respiratory syndrome coronavirus type 2 and its specific antibody. Background technique [0002] The novel coronavirus disease 19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged 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 were more than 14 million confirmed cases of COVID-19 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, existing research reports and data show that the lethality rate of SARS-CoV-2 ...

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 金梅林杨丽黄坤陈焕春涂亚平回显锋陈洁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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