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A kind of anti-HIV monoclonal antibody and its preparation method and application
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A monoclonal antibody and variable region technology, applied in the preparation of anti-viral infection monoclonal antibodies, the preparation of anti-infective drugs, the preparation of monoclonal antibodies, and can solve the problems of viral load rebound, limited efficacy, and limitations , to achieve the effect of improving the amplification efficiency
Active Publication Date: 2022-04-26
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI +1
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[0004] However, it is worth noting that the broad-spectrum high-efficiency neutralizing antibody still has relatively limited efficacy in the treatment of established infections. In patients receiving bNAbs treatment, due to the possible existence of escape strains in advance, or the single antibody against The selection pressure of the virus is relatively small, so that after the antibody is used for a period of time, most patients will soon have a viral load rebound due to the development of the mutant virus
At the same time, once the infection is established, the ability of neutralizing antibodies to block the virus from cell-to-cell transmission will also be significantly reduced
In addition, bNAbs currently isolated are mainly from patients in North America and Africa. The prevailing strains are mainly subtype B and subtype C. The main epidemic strains in my country are mainly subtype CRF01-AE and subtype CRF07_BC. -AE infected patients have the characteristics of high viral load and rapid disease progression, the application of existing bNAbs is affected and limited, and the efficacy against the epidemic strains and infected patients in China will also be limited
In addition, the current human anti-HIV monoclonal antibody preparation technology has limited efficiency in the amplification of antibody genes.
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Embodiment 1
[0027] Embodiment 1: The preparation process of the anti-HIV monoclonal antibody is described in detail.
[0029] 1. Sample: This study passed the review of the Ethics Committee of Peking Union Medical College Hospital, and all patients signed the informed consent.
[0030] Inclusion criteria:
[0031] (1) The results of the primary screening test and Western blot detection of HIV antibodies are all positive;
[0032] (2) HIV infection for more than 4 years;
[0033] (3) Not receiving antiretroviral treatment;
[0034] (4) The plasma viral load can be detected;
[0035] (5) There are PBMC samples.
[0036] Exclusion criteria:
[0037] (1) Age older than 65 or younger than 18;
[0038] (2) Combined with severe opportunistic infection causing failure of important organ function;
[0039] (3) Severe liver and kidney disease;
[0040] (4) Patients with active tumors;
[0041] (5) Pregnant and lactating patients.
[004...
Embodiment 2
[0252] Example 2: The monoclonal antibody 7D5 obtained in Example 1 is used to inhibit HIV infection
[0253] 1. Virus inhibition experiment
[0254] (1) Cell preparation: TZM-bl cells were subcultured at least three times after recovery, and the cell viability was above 90%.
[0255] (2) In a 96-well cell plate, 50 μl (200TCID 50 ) HIV (REJO4541.67) was incubated with 100 μl of different concentrations of monoclonal antibody 7D5 at 37° C. for 1 h.
[0256] (3) Immediately add 100μl concentration of 1.3×10 5 cells / ml of TZM-bl cells, DEAE-dextran hydrochloride has been added to the cell suspension, and its final concentration is 10 μg / ml.
[0257] (4) Cultivate at 37°C with 5% CO2 for 48 hours.
[0258] (5) Reading: use a microplate reader to detect the fluorescent signal in the well plate.
[0259] (6) Calculate the virus inhibitory activity of the antibody: inhibition rate=(V-T) / (V-C), where T is the sample to be tested, C is the cell control, and V is the virus control...
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Abstract
The invention discloses an anti-HIV monoclonal antibody, its preparation method and application. The monoclonal antibody includes a heavy chain and a light chain, specifically as shown in the sequence listing. In the present invention, YU2 gp140 trimer and AE1 gp140 trimer double antigens are used to sort specific single memory B cells from long-term non-progressors infected with HIV, obtain paired antibody heavy chain and light chain genes, and screen to obtain anti-HIV single Cloned antibody 7D5. The antibody has high binding activity and limited neutralizing activity. The optimized antibody gene cloning protocol improves the amplification efficiency of heavy and light chains. The discovery of this monoclonal antibody not only provides an alternative drug for the prevention and treatment of HIV infection, but also provides a technical reference for the development of monoclonal antibody drugs against viral infection.
Description
technical field [0001] The invention belongs to the technical field of antiviral infection monoclonal antibody preparation, in particular to a monoclonal antibody capable of effectively inhibiting human immunodeficiency virus (human immunodeficiency virus, HIV) infection. At the same time, the present invention also relates to the preparation method of the monoclonal antibody, and the application of the antibody in the preparation of anti-infective drugs. Background technique [0002] HIV is an infectious CD4 + Immune cell lentiviruses, a genus of retroviruses, were first discovered in Central America in 1981. HIV infection can lead to acquired immune deficiency syndrome (Acquired Immune Deficiency Syndrome, AIDS). There is currently no effective vaccine to prevent HIV infection, and the virus is almost incurable once infected. There are currently 36.9 million infected cases in the world, about 1.7 million new cases each year, and 770,000 people die from AIDS. There are ...
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