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GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein and preparation method thereof

A GBM-7S, fusion protein technology, applied in the field of antigen preparation, can solve problems such as poor antigenicity of GBM antigens

Active Publication Date: 2020-05-01
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first purpose of the present invention is to provide a GBM-7S(α1)-NC1(α3) fusion protein to alleviate the problem of poor antigenicity of GBM antigens in the prior art and urgent need for new antigens

Method used

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  • GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein and preparation method thereof
  • GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein and preparation method thereof
  • GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein and preparation method thereof

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Experimental program
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preparation example Construction

[0051] In a preferred embodiment, the preparation method of nucleotide fragments comprises:

[0052] (a) performing PCR amplification on the 7S domain of the human COL4α1 gene to obtain a PCR product;

[0053] (b) performing PCR amplification on the NC1 domain of the human COL4α3 gene to obtain a PCR product;

[0054] (c) Ligate the PCR product in (a) with the PCR product in (b) to obtain a nucleotide fragment.

[0055] In a preferred embodiment, the signal peptide of amino acid 1-27 is removed from the PCR product in (a), and the GP67 signal peptide is added at the 5' end. Optimizing the signal peptide is beneficial to the secretion of the fusion protein and subsequent protein purification.

[0056] In a preferred embodiment, a 6his-tag tag is added to the 3' end of the PCR product in (b). Adding a protein tag to the fusion protein facilitates subsequent protein purification.

[0057] In a preferred embodiment, the vector includes an expression vector, preferably a pFastB...

Embodiment 1

[0059] Secretion expression method of embodiment 1GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein

[0060] (1) The 7S domain of the human COL4α1 gene is amplified by PCR to obtain a PCR product, the signal peptide at amino acid 1-27 positions is removed, and the GP67 signal peptide is added at the 5' end. The amino acid sequence of the GP67 signal peptide is as follows: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA (SEQ ID NO.2);

[0061] (2) Perform PCR amplification on the NC1 domain of the human COL4α3 gene. The 5' end primer is ggctttgtcttcacccgac (SEQ ID NO.3), and the 3' end primer is tcttttcttcatgcac (SEQ ID NO.4). Obtain the PCR product;

[0062] (3) The above two gene fragments were sequentially connected and inserted between the BamHI and HindIII sites of the vector pFastBac1, and a 6his-tag tag was added to the 3' end, and the recombinant fragment was GP67-7S(α1)-NC1(α3) -6histag, construction of expression plasmid GP67-7S(α1)-NC1(α3)-6histag;

[0063] (4) Transform the ...

Embodiment 2P1

[0064] The preparation of embodiment 2P1 generation virus

[0065] (1) Inoculate about 900,000 Sf9 cells (in the logarithmic growth phase and with a viability greater than 95%) in a six-well plate, and culture at 27° C. for 1 hour to allow the cells to adhere to the wall.

[0066] (2) After the cells are completely adhered to the wall, the medium is aspirated, and the cells are washed with 1.5 ml of medium (SF900-III, no antibiotics and no serum) to remove residual antibiotics.

[0067] (3) Add 1.5ml medium (SF900-III, no antibiotics and no serum) to continue culturing until transfection.

[0068] (4) Preparation of Bacmid DNA and transfection reagent mixture.

[0069] ① Take a 1.5ml sterile centrifuge tube, dilute 16μg of Bacmid DNA into 100μl of SF900-III medium (no antibiotics and no serum), gently blow and mix with a gun, and let stand at room temperature for about 2-5min;

[0070] ② Take another 1.5ml sterile centrifuge tube, absorb 8μl of transfection reagent and dilut...

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Abstract

The invention relates to the field of antigen preparation, and particularly provides a GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein and a preparation method thereof. The invention provides a GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein with an amino acid sequence as shown in SEQ ID NO.1. The fusion protein has good antigenicity, and can be effectively subjected to recognition reaction by a humanGBM antibody. The preparation method of the GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein comprises the following steps: transfecting insect cells with a recombinant vector constructed from a vectorand a nucleotide fragment provided by the invention, and then carrying out protein expression to obtain the GBM-7S (alpha 1)-NC1 (alpha 3) fusion protein. By the method, a large amount of GBM antigenswith good antigenicity can be stably expressed by utilizing the insect cells; the method is simple and easy to operate; and biological modules obtained in the whole process can be independently preserved and applied.

Description

technical field [0001] The invention relates to the field of antigen preparation, in particular to a GBM-7S(α1)-NC1(α3) fusion protein and a preparation method thereof. Background technique [0002] Glomerular basement membrane protein (glomerular basement membranes, GBM) is the target antigen of anti-GBM antibodies, and its main antigenic site is located in the glomerular basement membrane type IV protein α3 chain (Collagen alpha-3(IV) chain, hereinafter referred to as COL4α3) The NC1 domain in . Obtaining natural GBM protein needs to be extracted from the kidney, which is very difficult to extract and has a low yield, so the expression of recombinant GBM protein is very valuable. [0003] Human globular basement membrane (GBM) type IV protein mainly consists of six subtypes: α1, α2, α3, α4, α5 and α6 six subtypes. Its main antigenic site is located in the NC1 domain in the α3 subtype. The GBM protein is mainly composed of the N-terminal 7s domain, the C-terminal NC1 dom...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N5/10C07K16/18
CPCC07K14/47C07K16/18C07K2319/00C07K2319/02C07K2319/21C12N5/0601C12N15/86C12N2510/02C12N2710/14043C12N2800/105
Inventor 杨滨王丽高雪丹黄卓春张君龙胡静
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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