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Nanometer antibody synthesis library and construction method thereof

A nanobody and construction method technology, applied in the field of genetic engineering, can solve the problems of low antibody affinity, long library construction period, and high storage capacity requirements, and achieve high expression and good thermal stability.

Pending Publication Date: 2022-07-29
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the antibody of the immune library has high affinity and strong specificity, and only needs a small storage capacity (10 6 ) to prepare antibodies, but the construction period of the immune library is long (1.5-3 months), requires animal immunization, and is not suitable for highly toxic / non-immunogenic objects; the natural library is rich in diversity and only needs a small amount of antigen panning Screening can be used as a broad-spectrum preparation platform, but the natural library requires a large number of different individual animal blood, generally the antibody affinity obtained is low, the diversity is limited by V-D-J rearrangement, and the library capacity requirements are high (>10 8 ); the diversity of the synthetic library is rich, only a small amount of antigen panning is required, and it can be used as a broad-spectrum preparation platform with high storage capacity requirements (>10 8 )

Method used

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  • Nanometer antibody synthesis library and construction method thereof
  • Nanometer antibody synthesis library and construction method thereof
  • Nanometer antibody synthesis library and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Determination of backbone and randomization site

[0041] In the present invention, a conserved framework region of a nanobody with high expression and high thermal stability is used as the framework of the synthetic library. The expression amount of the framework is 30-50 mg / L, and it can still maintain 50% when heated at an extreme temperature of 90°C for 60 minutes. activity (Wang J, Bever C, Majkova Z, et al. Heterologous antigen selection of camelidheavy chain single domain antibodies against tetrabromobisphenol A. [J]. Analytical Chemistry, 2014, 86(16):8296-8302.); this framework phase Compared with the general framework, the expression amount is nearly an order of magnitude higher, with high expression amount and good thermal stability. Randomized fragments of the synthetic library are designed based on their DNA sequences for synthetic library construction and subsequent panning.

[0042] The selected framework gene sequence was uploaded to this webs...

Embodiment 2

[0046] Example 2 Amplification of synthetic gene fragments

[0047] According to the determined FR regions and CDRs regions, four single-stranded DNA fragments with randomization sites were synthesized. CDR3-FR4 part, and design corresponding extension primers to amplify single-stranded DNA into double-stranded DNA.

[0048] The antibody gene sequence was designed with four single-stranded DNA fragments and their matching extension primers as shown in Table 1 below. Using single-stranded DNA as a template, the corresponding double-stranded DNA fragments were obtained by Klenow enzyme amplification, and the reaction of amplifying the double-stranded DNA fragments System and conditions: First, the molar ratio of single-stranded DNA and extension primers was controlled to be 1:3, then the system was supplemented to 50 μL with TE buffer (containing 100 mM NaCl), heated at 95 °C for 5 min, and slowly cooled at room temperature for 20 min. Add 20 μL of 10×NEBbuffer2, 8 μL of 10mM d...

Embodiment 3

[0053] Embodiment 3 Optimization of overlapping extension PCR reaction conditions

[0054] The randomized regions of the synthetic library constructed by the method of the present invention are designated as CDR1, CDR2 and CDR3 regions, and all three CDR regions are randomized simultaneously. When randomizing the region selection, generally the more randomized sites are, the more complex and difficult the amplification reaction will be. Selecting all CDR regions for randomization requires multiple extension and splicing, which is relatively more difficult. In this example, the overlapping extension PCR reaction conditions and primers are optimized, wherein a series of specific primers are designed for the splicing of the Nanobody gene sequence as shown in Table 2 below, and the overlapping extension PCR reaction conditions are optimized as shown in Table 3 below.

[0055] The antibody gene fragments obtained in the above steps were spliced ​​by overlap extension PCR. The overl...

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Abstract

The invention discloses a nano antibody synthesis library and a construction method thereof. The construction method comprises the following steps: selecting a nano-antibody skeleton region with high expression quantity and high thermal stability as a basic antibody framework of a synthetic library, then determining a randomized region through nano-antibody sequence collection and data statistical analysis, adopting an NNK degenerate codon to introduce sequence diversity in a randomized strategy, and amplifying a synthetic fragment through a Klenow enzyme, so as to obtain the nano-antibody with high expression quantity and high thermal stability. A complete nanometer antibody sequence containing randomized sites is obtained after multiple times of overlapping, extending and splicing, and the nanometer antibody sequence is used for constructing a nanometer antibody synthesis library with high capacity and high diversity. Wherein the expression quantity of the framework is greater than 20mg / L, and the thermal stability means that the framework can still maintain activity close to room temperature after being heated at 50-90 DEG C for 5-60 minutes; according to the present invention, the CDR3 regions with three lengths are designed, and the three CDR regions are randomized, such that the three nanometer antibody synthesis libraries with different lengths are finally constructed, and the sequence diversity and the structure diversity of the libraries are increased;

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, it relates to a nanobody synthesis library and its construction method. Background technique [0002] Phage display technology integrates foreign genes into specific phage genes through genetic engineering technology, and finally displays the active target protein through the phage coat protein. Common techniques. [0003] The relative molecular mass of nanobodies (VHH) is 12-15kDa, which is only one tenth of that of traditional monoclonal antibodies. This makes it easy to access grooves, fissures, or hidden epitopes on the surface of the target, recognizing many antigens that traditional antibodies cannot. The acquisition of nanobodies needs to be screened through the nanobody library. At present, the nanobody library can be divided into three types according to their sources, namely natural library, immune library and synthetic library. The Nanobody libraries ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/10C12N15/13C12N15/70C12N15/66C07K16/00
CPCC40B50/06C40B40/10C12N15/70C12N15/66C07K16/00C07K2317/565C07K2317/567C07K2317/569C12N2800/101
Inventor 王弘刘飞张奇李迎雪
Owner SOUTH CHINA AGRI UNIV
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