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Method for rearranging immunoglobulin and producing antibody through MDA-PCR (Multiple Displacement Amplification-Polymerase Chain Reaction) enriched genome

An immunoglobulin and genome technology, applied in biochemical equipment and methods, genetic engineering, DNA preparation, etc., can solve problems such as substrates that are not suitable for PCR amplification of target genes, avoid mRNA instability, and reduce subsequent pollution. , the effect of simple steps

Active Publication Date: 2014-01-22
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNA fragments amplified in this way are not suitable as substrates for PCR amplification of target genes due to the wide variety of sequences.

Method used

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  • Method for rearranging immunoglobulin and producing antibody through MDA-PCR (Multiple Displacement Amplification-Polymerase Chain Reaction) enriched genome
  • Method for rearranging immunoglobulin and producing antibody through MDA-PCR (Multiple Displacement Amplification-Polymerase Chain Reaction) enriched genome
  • Method for rearranging immunoglobulin and producing antibody through MDA-PCR (Multiple Displacement Amplification-Polymerase Chain Reaction) enriched genome

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Figure 5A The analyzes of lanes 1-5 and lanes 6-10 were for serial dilutions of mouse B cells (approximately 1000, 100, 10, 1, 0.1 cells). After cell lysis (step 1), VH (390-400bp) and Vk (370-380bp) genes were enriched with or without MDA (step 2), and then amplified by PCR (step 3), wherein lane 1- 5 performed PCR only, lanes 6-10 performed MDA-PCR as Figure 5A As shown, the VH and Vk genes can be simultaneously amplified in the reaction tube with only a single cell in each tube (channel 9), and if no MDA enrichment is carried out, the IgH gene can be detected only at the level of 100 and 1000 cells (channel 9). 2 and 1). in Figure 5A of channels 6-10 and Figure 5B The primers for MDA in lanes 6-10 are all the specific primers in Table 2. Figure 5A Lanes 1-5 are the results of direct PCR without MDA amplification, Figure 5B Lanes 1-5 are the results of PCR after MDA amplification with random hexamer primers. exist Figure 5B Lanes 6-10 show MDA enrichmen...

Embodiment 2

[0076] Example 2 is the isolation of mouse antibody genes from single B cells by MDA-PCR. Using fluorescence-activated cell sorting (FACS), single mouse κ memory B cells were distributed in 96-well plates and then enriched for MDA. respectively by IgH( Figure 6 , upper part) or Igk ( Figure 6 , lower) primers for further PCR amplification of MDA-enriched genomic DNA. Figure 6 Including upper and lower glue, the channel is corresponding. The upper gel shows the heavy chain (IgH) PCR amplification results of samples 1-24, and the lower gel shows the light chain (Igk) PCR amplification results of samples 1-24. The heavy and light chains of samples 1, 6, 10, 11, 12, 17, 20, and 21 (the samples indicated by the arrows) all obtained amplified target fragments, indicating that the samples were successfully amplified with complete antibodies Genes (both heavy and light chains), those samples where only one chain was amplified, were not actually able to obtain a complete antibod...

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Abstract

The invention provides a method for rearranging immunoglobulin by specifically amplifying a genome. The method comprises the following steps of: cracking mature B cells; amplifying genome genes of the B cells through a primer capable of specifically amplifying a variable region of a heavy chain and / or a light chain of an antibody by using a multiple displacement amplification method, thereby amplifying a sequence of the variable region comprising the heavy chain and / or light chain from the genome relatively and specifically; and performing PCR amplification by using the primer for specifically combining the variable region of the heavy chain and / or the light chain of the antibody by taking reaction solution obtained by multiple displacement amplification as a substrate, thereby obtaining a gene sequence of the variable region of the heavy chain and / or the light chain of the antibody; sequencing an obtained PCR product to determine the gene sequence of the variable region of the heavy chain and / or the light chain of the antibody obtained by PCR amplification. Through the method provided by the invention, rearranged immunoglobulin is obtained and the gene is utilized to product the antibody.

Description

technical field [0001] The present invention relates to a new method for antibody production, more specifically, relates to a method for enriching genome rearrangement immunoglobulin gene by MDA-PCR and using the gene for antibody production. Background technique [0002] The humoral immunity of animals is mainly accomplished through antibodies secreted by B cells. Such as figure 1 As shown in , an antibody consists of four polypeptide chains, of which the two longer, relatively larger molecular weight chains are heavy chains (also called H chains); the two shorter, relatively smaller molecular weight chains are light chains (also known as H chains). called the L chain). The chains are linked by disulfide bonds and non-covalent bonds. There are two types of light chains, κ and λ, and five types of heavy chains, μ, δ, γ, ε, and α. The whole antibody molecule can be divided into two parts, the constant region (also known as the C region) and the variable region (also known...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C12N15/10C12N15/79
Inventor 蔡槱伯
Owner BERRYGENOMICS CO LTD
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