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Taq DNA polymerase single-chain immunoglobulin IgG antibody, and preparation method and application thereof in genotyping detection

A technology of immunoglobulin and single-chain antibody, which is applied in the direction of anti-enzyme immunoglobulin, immunoglobulin, recombinant DNA technology, etc., can solve the problems affecting the specificity of the reaction, the amplification of non-target sequences, etc., and achieve improved efficiency and high efficiency. Effects of functional stability, simplified preparation procedures, and antibody structure

Pending Publication Date: 2019-09-20
江苏苏博生物医学科技南京有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The result can lead to the amplification of non-target sequences, affecting the specificity of the reaction

Method used

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  • Taq DNA polymerase single-chain immunoglobulin IgG antibody, and preparation method and application thereof in genotyping detection
  • Taq DNA polymerase single-chain immunoglobulin IgG antibody, and preparation method and application thereof in genotyping detection
  • Taq DNA polymerase single-chain immunoglobulin IgG antibody, and preparation method and application thereof in genotyping detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Expression of scFv in prokaryotic cells:

[0038] Take out the centrifuge tube containing the competent cells of Rosetta-gami 2 (DE3) strain from -80°C, quickly insert it into ice, wait for the bacterial block to melt after 5 minutes, and add the recombinant plasmid DRSU02-2 pET28a- VL-(Gly4S)3-VH-HIS6 and DRSU032-1 pET28a-VH-218-VL-HIS6, mix gently (avoid pipetting with a gun), and let stand in ice for 25 minutes. Subsequently, the transformation system was heat-shocked in a water bath at 42° C. for 45 seconds, quickly put back on ice and allowed to stand for 2 minutes. Add 200 μl of sterile LB medium without antibiotics to the centrifuge tube, mix well and recover at 37°C and 200rpm for 60 minutes. Spread the recovered bacterial liquid onto LB plates with 50 μg / mL kanamycin (Kan). Place the plate upside down and incubate overnight in a 37°C incubator. Single-bacteria pick-point activation culture was carried out on the plates cultured overnight to obtain recombinan...

Embodiment 2

[0041] 1. Detection principle of hot start Taq enzyme effect

[0042] Use 1 pair of primers that anneal at room temperature to form a 3 bp overlap. If the DNA polymerase extends the primer prior to heat activation, an 82 bp primer-dimer will be formed which can serve as a template for amplification in subsequent cycles. However, efficient hot-start enzymes do not generate primer-dimers because enzyme activation and subsequent PCR cycling temperatures are higher than the annealing temperature of the 3 bp overlap region. Based on this, the hot start effect of the polymerase at room temperature can be judged, and the optimal antibody concentration can be selected. No genomic DNA template is required in this simple assay format.

[0043] 2. Hot start Taq enzyme preparation and effect detection

[0044] The antibody prepared in Example 1 was serially diluted to obtain 2×, 4×, 8×, and 16× dilutions. The diluents of each concentration were mixed with equal volumes of 10 U / μl Taq ...

Embodiment 3

[0052] Example 3: Application on STR Genotyping

[0053] Hot-start Taq DNA polymerase was prepared according to Example 2. Mix equal volumes of 1× antibody with 10 U / μl Taq enzyme to obtain 5 U / μl hot-start Taq DNA polymerase. According to the Subo Yansu Y41SE STR detection kit, the reaction system was established as follows:

[0054]

[0055] And carry out amplification on the PCR instrument according to the following parameters:

[0056]

[0057] After the PCR amplification, the DNA was denatured, and electrophoresis was performed on a capillary electrophoresis instrument. The results were as follows: Figure 11-12 shown. Figure 11 It shows that the amplification uniformity of the Taq DNA polymerase site without adding single-chain antibody is poor, and the amplification of individual sites fails. And the hot-start Taq DNA polymerase ( Figure 12 ) can amplify all sites, and the uniformity between sites is greatly improved compared with non-hot start Taq DNA poly...

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Abstract

The invention discloses a Taq DNA polymerase single-chain immunoglobulin IgG antibody, and a preparation method and an application thereof in genotyping detection. The Taq DNA polymerase single-chain immunoglobulin IgG antibody has the amino acid sequence consisting of a heavy chain variable region, a connecting peptide and a light-chain variable region. The connecting peptide is located between the heavy chain variable region and the light chain variable region. The Taq DNA polymerase single-chain immunoglobulin IgG antibody not only has high functional stability, but also simplifies the preparation procedures and the antibody structure, improves the preparation efficiency, improves the potency of the antibody, and can be used as a monoclonal antibody for preparing a hot start enzyme, or as a neutral antibody of a binding target to realize the function of inhibiting the binding target.

Description

technical field [0001] The invention relates to a Taq DNA polymerase single-chain immunoglobulin IgG antibody, a preparation method and its application in genotyping detection, belonging to the field of genetic engineering. Background technique [0002] Today, antibodies are used more and more widely, including detecting the expression of related antigens and producing targeted drugs. The industrial production of antibodies mainly relies on the purification of the supernatant of hybridoma cells that secrete the corresponding antibodies, but it is difficult to obtain the hybridoma cells corresponding to the antibodies in this process, so this method of antibody preparation is difficult to be promoted. Genetic engineering is used to make the cells express the heavy chain and light chain of the corresponding antibody, and after heterologous expression in the host cell, the antibody is obtained through a certain purification method. This method is easy to operate and popularize....

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/70C12P21/02C12N9/12C12N9/96C12Q1/6858
CPCC07K16/40C12N15/70C12N9/1252C12N9/96C12Q1/6858C12Y207/07007C07K2317/622C07K2317/76C12Q2521/101C12Q2531/113C12Q2527/125
Inventor 苏雪峰臧颖朱凯张明航
Owner 江苏苏博生物医学科技南京有限公司
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