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Method for quantitative PCR detection of exogenous gene copy number in transgenic cell

A technology of transgenic cells and copy number, applied in the field of molecular biology, can solve the problem that the original DNA copy number cannot be calculated, and achieve the effect of accurate determination of gene copy number, high specificity and good linear relationship.

Inactive Publication Date: 2016-12-07
GENOR BIOPHARMA
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Problems solved by technology

However, in the late stage of PCR amplification, with the continuous increase of products, the amplification products no longer increase exponentially, and there is no linear relationship between the amount of final PCR products and the amount of initial template, and the starting template cannot be calculated based on the amount of final PCR products. original DNA copy number

Method used

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  • Method for quantitative PCR detection of exogenous gene copy number in transgenic cell
  • Method for quantitative PCR detection of exogenous gene copy number in transgenic cell
  • Method for quantitative PCR detection of exogenous gene copy number in transgenic cell

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Embodiment 1

[0045] The quantitative PCR detection method of embodiment 1 light chain gene copy number

[0046] 1.1. Plasmid DNA acquisition:

[0047] a) Design specific primers for the light chain gene: the nucleotide sequence of the specific forward primer JLc1F is: 5'-AGGACAGTGGCTGCACCA-3' (as shown in SEQ ID No.5); the specific reverse primer JLc1R The nucleotide sequence is: 5'-TCAACACTTCCTCTGTT-3' (as shown in SEQ ID No. 6).

[0048] b) Obtain the light chain pLc plasmid strain: use the light chain DNA of the complete antibody molecule as a template, perform ordinary PCR amplification to obtain the light chain Lc fragment of the antibody molecule, and then insert it into the psimple-19T vector to transform DH5α competent cells , obtained the pLc plasmid strain containing the antibody light chain gene.

[0049] c) After sequence determination, the pLc plasmid strain with a molecular size of 3016bp was obtained.

[0050] 1.2. Preparation of light chain plasmid DNA standard

[0051]...

Embodiment 2

[0079] Example 2 Detection method of heavy chain gene copy number

[0080] 2.1. Plasmid DNA acquisition:

[0081] a) Design the forward primer JHc-1F and the reverse primer JHc-1R for the heavy chain gene:

[0082] The nucleotide sequence of the forward primer JHc-1F is: 5'-ATGCTAGCAAGCTTCCATGGGCGTCGACAAAGGGACCATCTGTGT-3' (as shown in SEQ ID No.7); the nucleotide sequence of the reverse primer JHc-1R is: 5'-TAGGTACCTCGAGTCATTTACCAGGAGACAG-3 ' (as shown in SEQ ID No.8).

[0083] b) Obtain heavy chain pHc plasmid strain

[0084] Using the heavy chain DNA of the complete antibody molecule as a template, carry out ordinary PCR amplification to obtain the heavy chain Hc fragment of the antibody molecule, and then insert it into the T vector to transform DH5α competent cells to obtain the heavy chain pLc plasmid strain.

[0085] c) After sequence determination, a pHc plasmid strain with a molecular size of 3718bp was obtained.

[0086] 2.2. Preparation of heavy chain standard pl...

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Abstract

The invention relates to a method for quantitative detection of the light and heavy chain gene copy numbers of antibody molecules in a transgenic animal cell CHO. An SYBR-Green quantitative PCR method is adopted to realize quantitative, accurate and high-flux detection of the light chain gene copy number and the heavy chain gene copy number of the antibody molecules. The method has the advantages of high detection sensitivity, wide linear range and reliable result, can be used for researching the stability of antibody cell strains and screening stable high-expression engineering cell strains.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a quantitative detection method for the copy numbers of light and heavy chain genes of antibody molecules in transgenic animal cells. Background technique [0002] Animal cells have been widely used in the production of various biologically active substances such as vaccines, growth factors, antigens, monoclonal antibodies and other recombinant protein drugs. These animal cells include 293 cells, Vero cells, BHK cells, PER.C6 cells, SP2 / 0 Cells, NSO cells, CHO cells, etc., among which the most widely used is CHO cells. There are more than 20 kinds of recombinantly expressed antibody drugs approved by the US FDA, creating a market worth tens of billions of dollars each year. With the development of genetic engineering technology, more and more foreign proteins such as antibodies are obtained through transgenic methods. [0003] However, for transgenic cells, exogenous ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 薛建华黄雪怡徐水清
Owner GENOR BIOPHARMA
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