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Potential preparation method for highly-efficient recombinant HIV-1 CRF07-BC gp140 immunogen

A CRF07, HIV-1 technology, applied in the field of bioengineering, to achieve the effect of high antigen reactivity and good uniformity

Inactive Publication Date: 2014-08-20
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this trimer has a disadvantage. Although the introduction of disulfide bonds plays a very important role in the process of stabilizing the interaction between gp120 and gp41 ectodomain, it may also lock this gp140 in the pre-fusion conformation (the gp140 Crystal and electron microscope structures support this view), as an immunogen is not necessarily conducive to the production of broad-spectrum neutralizing antibodies

Method used

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  • Potential preparation method for highly-efficient recombinant HIV-1 CRF07-BC gp140 immunogen
  • Potential preparation method for highly-efficient recombinant HIV-1 CRF07-BC gp140 immunogen
  • Potential preparation method for highly-efficient recombinant HIV-1 CRF07-BC gp140 immunogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Construction of recombinant gp140 eukaryotic expression vector

[0018] Using the CRF07-BC gp160 cDNA as a template, two overlapping extension PCRs were performed to obtain the extracellular domain of the envelope glycoprotein Env linked with two different linkers (see figure 1 ) DNA fragment gp140, the target gene fragment was cloned into the pMT / Bip / TEV-HisA vector modified by our laboratory by the method of double digestion and ligation. By replacing the V5 epitope with the cleavage site of tobacco etch virus protease (TEV enzyme), we can use TEV enzyme to remove the histidine tag (6×His-tag) at the carboxyl terminus of the target protein, so that the carboxyl group of the target protein can be removed. End with as few unnecessary oligopeptides as possible. After double-enzyme digestion and sequencing identification (see the sequence table for amino acid and nucleotide sequences), the target plasmid is extracted by an endotoxin-free plasmid extraction kit...

Embodiment 2

[0026] Example 2. Co-transfection of S2 cells and screening of S2 cell lines stably expressing target proteins

[0027] Cells were plated in T25 culture flasks one day in advance, and co-transfection was performed by liposome method when the confluency of cells reached 60-70%. The transfection step was carried out according to the instructions of the transfection reagent Cellfectin II, and it was noted that the mass ratio of the recombinant plasmid and the resistance screening plasmid pCoBlast was 19:1. After the cells were incubated at 27°C for 5 hours, the transfection solution was aspirated, 4 mL of fresh SFX-Insect medium was added, and the incubation was continued for 48 hours. The original medium was discarded, and SFX containing blasticidin (final concentration of 25ug / mL) was added. -Insect medium for resistance screening, unsuccessfully transfected cells will die a lot within a week after adding blasticidin for screening, and a small number of adherent cells will cont...

Embodiment 3

[0028] Example 3. Expression and purification of recombinant gp140

[0029] The selected T25S2 cell line was expanded into a T75 culture flask (volume ratio 1:5), and further transferred to a 500mL spinner flask for expanded culture (volume ratio 1:10), 27°C, 120rpm, and cultured for 2-3 days , when the growth of cells reached logarithmic phase, copper sulfate was added to induce protein expression (final concentration was 0.5 mmol / L), and the cells were collected after 3 days. The S2 cells were collected into a 300 mL centrifuge bucket, and centrifuged at 4000 rpm for 15 min at 4°C. The cell supernatant was collected, filtered through a 0.22 μm filter, and placed in an Amicon Stirred Cell8003 ultrafiltration cup to concentrate the protein. When compressed to 50 mL, the cells were combined with a nickel column binding buffer (50 mM / L Tris-HCl, 500 mM / L NaCl, After diluting 10 times with pH 8.0), the protein solution was collected into a 50 mL centrifuge tube, centrifuged at 2...

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Abstract

The invention discloses a potential preparation method for a highly-efficient recombinant HIV-1 CRF07-BC gp140 immunogen. The immunogen is designed based on the structure of HIV-1 envelope protein crystals which have been published internationally and obtained in our lab. The specific method uses an overlap extension PCR technology to obtain gp140 gene segments, and comprises the steps that target genes are cloned into an eukaryotic expression vector pMT, endotoxin is removed through extraction of a large amount of plasmids, the gp140 gene segments and resistance screening plasmids pCoBlast are co-transfected into drosophila melanogaster Schneider2 (S2) cells together, blasticidin (Blasticidin S) is used for positive clone screening, S2 cell lines for stably and efficiently secreting and expressing gp140 are screened, and after enlarged cultivation and through two steps of purification of nickel column affinity chromatography and gel filtration chromatography, the gp140 with high purity can be obtained. A series of biochemical and biophysical technologies indicate that the gp140 is uniform in polymeric states, and high in antigenic reactivity, and is quite fittingly used as the immunogen for the research and the development of AIDS subunit vaccines or multivalent combined vaccines.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, and specifically relates to immunogen design based on protein structure, molecular cloning, liposome transfection, protein expression and purification, analytical ultracentrifugation (SV-AUC) and enzyme-linked immunosorbent assay (ELISA), etc. technology. Background technique: [0002] AIDS (accquired immunodeficiency syndrome, AIDS) is caused by human immunodeficiency virus (human immunodeficiency virus, HIV) infection caused by a high fatality rate is a chronic infectious disease raging around the world. HIV is divided into two types: HIV-1 and HIV-2. HIV-1 is more virulent and infectious than HIV-2, and causes AIDS worldwide. HIV-2 is basically only found in parts of West Africa. . According to data from the World Health Organization website, as of October 2013, the number of HIV-infected people worldwide was 36 million, and the cumulative number of deaths exceeded 36 million. In 2012, there w...

Claims

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Application Information

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IPC IPC(8): C07K14/155C12N15/85C07K1/22C07K1/16
CPCC07K14/16
Inventor 刘新奇段良伟
Owner NANKAI UNIV
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