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ProA/G-dRep fusion protein serving as universal carrier of nucleic acid-antibody codons and application of ProA/G-dRep fusion protein

A fusion protein and antibody technology, applied in the field of genetic engineering, can solve the problems of uncontrollable coupling quantity, uncontrollable coupling quantity, antibody inactivation, etc.

Active Publication Date: 2021-05-18
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used connection method between nucleic acid and antibody mainly relies on the covalent connection method of chemical coupling agent. The commonly used method is as follows: 1. Under the action of chemical coupling agent EDC, the carboxyl group and amino group of antibody and nucleic acid are coupled, The connection between nucleic acid and antibody is completed, but this method cannot control the position of coupling, which may lead to inactivation of antibody; 2. Modification of nucleic acid with maleimide group and coupling with free sulfhydryl group on single-chain antibody, Complete the connection of nucleic acid and antibody, this method cannot control the coupling position and the coupling quantity; 3. Use Thermofisher's commercial kit SiteClick TM The Antibody Azido Modification Kit azides the sugar chain at the Fc end of the antibody, and then performs a coupling reaction with the nucleic acid modified with DBCO at the 5' end to complete the connection between the nucleic acid and the antibody. Although this method can control the coupling position, it needs to introduce expensive Base modifications and other purification steps without control over the number of conjugations
It can be seen that the method in the prior art still has the disadvantages of high cost and the inability to determine the number of connections between the antibody and the single-stranded DNA

Method used

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  • ProA/G-dRep fusion protein serving as universal carrier of nucleic acid-antibody codons and application of ProA/G-dRep fusion protein
  • ProA/G-dRep fusion protein serving as universal carrier of nucleic acid-antibody codons and application of ProA/G-dRep fusion protein
  • ProA/G-dRep fusion protein serving as universal carrier of nucleic acid-antibody codons and application of ProA/G-dRep fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Selection of the amino acid sequence of the ProA / G-dRep fusion protein gene:

[0046] The amino acid sequence of duck circovirus replicase (Duck circovirus Rep) (Uniprot Accession No.A7LI84, SEQ ID No.4) is as follows:

[0047] MAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLNLRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEATSAV;

[0048] The fusion protein (ProA / G) amino acid sequence (SEQ ID No.2) of protein A / G is as follows:

[0049] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK;

[0050] The amino acid sequence (SEQ ID No.3) of flexible peptides is as follows:

[0051] GGGGSGGGGSGGGGS;

[0052] 2. ProA / G-dRep fusion protein amino acid sequence design

[0053] Place ProA / G at the N-terminus of the ProA / G-dRep fusion protein, place the flexible peptide between ProA / G and dRep, and place dRep at the C-terminus of the ProA / G-dRep fusion protein, and the result is as follows figure 1 As shown, the amino acid sequence is the ProA / G-...

Embodiment 2

[0059] 1. Preparation of ProA / G-dRep fusion protein genetic engineering bacteria

[0060] (1) Take 2 μL of the dissolved plasmid and add it to BL21(DE3) competent cells, and ice-bath for 10 minutes;

[0061] (2) After being heat-shocked in a water bath at 42°C for 90 seconds, immediately place it in an ice bath for 5 minutes;

[0062] (3) Add 900 μL of anti-LB medium, fix it horizontally in a shaker, shake at 37°C for 60 min;

[0063] (4) Discard 800 μL of the supernatant by centrifugation, resuspend the remaining bacterial solution and spread evenly on the LB plate containing 50 μg / ml kanamycin;

[0064] (5) Place it upside down in a constant temperature incubator and culture at 37°C for 12 hours to obtain the ProA / G-dRep fusion protein genetically engineered bacterium BL21(DE3) / pET-28a-ProA / G-dRep.

[0065] 2. Fermentation expression

[0066] (1) Take the well-grown monoclonal cells from the plate and transfer them to a test tube containing 5 mL of LB liquid medium (50 μg...

Embodiment 3

[0090] Ligation test of single-stranded deoxyribonucleic acid and ProA / G-dRep fusion protein

[0091] 1. Preparation of single-stranded deoxyribonucleic acid

[0092] (1) According to the literature (J.Am.Chem.Soc.2017, 139, 7030-7035), the specific single-stranded DNA sequence recognized by dRep is: 5'-AAG TA TTACCAGAAA-3' (SEQ ID No.5, referred to as dRep ssDNA for short, the underline represents dRep specifically recognizes and cleaves and connects the single-stranded deoxyribonucleic acid site, the molecular weight is about 4.655kD);

[0093] (2) Send the above deoxyribonucleic acid sequence to Shanghai Sangong for synthesis, add 100mM NaCl solution after synthesis to dissolve to a concentration of 100μM, and freeze at -30°C for later use.

[0094] 2. Single-stranded DNA ligation test

[0095] (1) Take ProA / G-dRep solution and dRep ssDNA to carry out the ligation reaction according to the table below, and the ligation reaction system is shown in Table 1:

[0096] Table...

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Abstract

The invention provides a ProA / G-dRep fusion protein serving as a universal carrier of nucleic acid-antibody codons and application of the ProA / G-dRep fusion protein, and particularly relates to the field of genetic engineering. The ProA / G-dRep fusion protein comprises a fusion protein of protein A / G, a flexible peptide and a duck circovirus replicase which are sequentially connected from the N end to the C end. The ProA / G-dRep fusion protein can accurately determine the connection number of antibodies and single-stranded DNA, is simple in synthesis method and can be widely applied to research of the nucleic acid-protein codons.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a ProA / G-dRep fusion protein as a universal carrier of nucleic acid-antibody conjugates and its application. Background technique [0002] Nucleic acid and protein are the two most important types of biological macromolecules that constitute the basis of life. At present, the commonly used connection method between nucleic acid and antibody mainly relies on the covalent connection method of chemical coupling agent. The commonly used method is as follows: 1. Under the action of chemical coupling agent EDC, the carboxyl and amino groups of antibody and nucleic acid are coupled, The connection between nucleic acid and antibody is completed, but this method cannot control the position of coupling, which may lead to inactivation of antibody; 2. Modification of nucleic acid with maleimide group and coupling with free sulfhydryl group on single-chain antibody, Complete the connection...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C12N1/21G01N33/72C12R1/19
CPCC07K14/31C12N9/127C12N15/70G01N33/72C12Y207/07048C07K2319/00G01N2333/805
Inventor 邢航沈林聂勇
Owner HUNAN UNIV
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