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HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof

A surface glycoprotein and mutant technology, which is applied in the field of antigen preparation, can solve the problem that the vaccine cannot be reproduced, achieve good assembly effect and increase the effect of expression

Active Publication Date: 2019-08-30
WUHAN J H BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the immunoglobulin-based vaccines are currently able to induce bNAbs compared to natural infection, and one of the reasons may be that these vaccines cannot reproduce the natural Env trimer

Method used

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  • HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof
  • HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof
  • HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Gene Amplification of HIV Mutant Surface Glycoprotein and Construction of Eukaryotic Expression Plasmid

[0026] 1. PCR amplification

[0027] (1) In this experiment, overlapping PCR was used to amplify the target fragment. Firstly, primers F1 and R2, F3 and R4 (as shown in Table 1) were used to amplify the target gene respectively; finally, primers F1 and R4 were used to amplify. Due to the introduction of two cysteine ​​mutations, the mutations are introduced twice to form a double mutation. The PCR reaction system is the same, and the extension time is 1-2 minutes according to the length of the target fragment.

[0028] Table 1

[0029]

[0030]

[0031] (2) The PCR reaction system of each round of PCR reaction is 50 μL, as shown in Table 2, the reaction conditions are: 95 ° C for 5 min; 32 cycles, the cycle reaction is: 95 ° C for 1 min, 55 ° C for 1 min, 72 ° C for 1- 3 min (depending on the length of the fragment); extend at 72°C for 10 min, and ...

Embodiment 2

[0051] Example 2 Insect Cell Expression System Purification of HIV Surface Glycoprotein

[0052] 1. Bacmid transfection of sf9 cells

[0053] 1-2 hours before transfection, sf9 cells were spread on 6-well cell culture plates, and the cell density grew to 80%. Take Bacmid (about 6-7μg) and 6μL transfection reagent cellfectin Add 100 μL of insect cell culture medium (Sf-900II SFM) to IIreagent, and let stand at room temperature for 5 minutes. Mix the two and let stand at room temperature for 35 minutes. Add 800 μL of medium to each well of a 6-well cell culture plate, then add the bacmid and transfection reagent mixture, and incubate at a constant temperature of 27°C. 6 hours after transfection, the transfection solution was aspirated, 2 mL of medium containing 1% penicillin-streptomycin was added, and the constant temperature culture was continued at 27°C.

[0054] 2. Expression and identification of target protein

[0055] 3-4 days after transfection, draw 1 mL of the vi...

Embodiment 3

[0061] Example 3 Determination of the expression level of HIV mutant surface glycoprotein Western Blot

[0062] 1. Determination of total protein concentration of samples by BCA method: BCA protein concentration determination kit was purchased from Beyontian Company. According to the number of samples, prepare an appropriate amount of BCA working solution with 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and mix well. Completely dissolve the protein standard, take 10 μL and dilute to 100 μL, so that the final concentration is 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the standard to the standard wells of the 96-well plate, and make up to 20 μL with the solution of the diluted standard. Add an appropriate volume of sample to the sample well of the 96-well plate, and make up to 20 μL with the diluted standard solution. Add 200 μL of BCA working solution to each well, and measure OD after standing at 37°C for 30 minutes 562 nm absorbance value. Afte...

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Abstract

The invention provides HIV mutant type surface glycoprotein and nanometer antigen and preparation method thereof. Lysine at the 195th site of the HIV mutant type surface glycoprotein is changed into cysteine, and threonine at the 427th site is changed into cysteine. The HIV mutant type surface glycoprotein is used for coating the surface of nanometer particles, and through assembly, the HIV mutanttype surface glycoprotein nanometer antigen is obtained. According to the HIV mutant type surface glycoprotein, in accordance with gp120 protein, cysteine mutation is performed, the tripolymer conformation before merging of the HIV virus surface glycoprotein gp120 is stabilized, and through the stabilization of the tripolymer conformation, the expression level is increased. The HIV mutant type surface glycoprotein and the ferritin are self-assembled to obtain the nanometer antigen granules, so that the nanometer antigen can be used for fast detection of patients suffering from HIV at the later period.

Description

technical field [0001] The invention relates to the technical field of antigen preparation, in particular to HIV mutant surface glycoproteins, nanometerized antigens and a preparation method thereof. Background technique [0002] Human Immunodeficiency Syndrome (AIDS) was first diagnosed in the United States in 1981. Since the human immunodeficiency virus (HIV), the causative agent of AIDS, was officially recognized in 1983, HIV has caused at least 60 million infected people worldwide, and at least 25 million infected people have died of the disease. Although the proportion of people infected with HIV has generally stabilized since 2000, the total number of people infected worldwide is still increasing year by year. The emergence and use of highly active antiretroviral therapy (HAART) has greatly reduced the emergence of new cases and greatly prolonged the survival time of infected people, making people seem to see the prospect of eradicating HIV. But so far, there is stil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C07K14/155C07K1/16C12N15/49G01N33/531C12R1/93
CPCC12N15/86C07K14/005G01N33/531C12N2710/14043C12N2740/16022C12N2740/16051G01N2333/155Y02A50/30
Inventor 彭贵青王健斌龚贻洲周军许俊峰李强
Owner WUHAN J H BIO TECH
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