IgG antibody affinity peptide-alkaline phosphatase fusion protein

A technology of fusion protein and phosphatase, which is applied in the field of bioengineering, can solve problems such as fusion label blockage, and achieve good effect, convenient use and high activity

Inactive Publication Date: 2011-01-19
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Theoretically, although alkaline phosphatase and antibody are fused and expressed, current research is mainly focused on the construction of single-chain antibody (ScFv) and AP fusion protein. However, it is necessary to prepare the corresponding fusion antibody for each antigen detection. Fusion tags are hindered when constructing antibody-enzyme fusion proteins

Method used

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  • IgG antibody affinity peptide-alkaline phosphatase fusion protein

Examples

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Effect test

Embodiment 1

[0059] Example 1 Construction of IgG antibody affinity peptide-alkaline phosphatase fusion protein gene expression vector

[0060] In order to link the alkaline phosphatase gene with the IgG affinity peptide gene, first provide restriction endonuclease cleavage sites upstream and downstream of the secreted alkaline phosphatase gene (secreted alkaline phosphatase, SEAP, GenBank Accession No: U35316) Spot Sac I and Pst I, amplify the alkaline phosphatase gene (1467bp) without N-signal peptide and C-terminal extended peptide by PCR, insert IgG affinity peptide downstream (Sac I and Pst I sites of pEZZ18) ; The second step provides a restriction endonuclease cutting site Not I at the upstream and downstream of the IgG affinity peptide-AP gene, and is amplified by PCR and inserted into the Not I site of pPIC9K to construct the Pichia pastoris expression vector pIgG Ab-AP / 9k, such as figure 1 shown.

Embodiment 2

[0061] Example 2 Electrotransformation of Pichia pastoris GS115

[0062] For transformation of pAb-AP / 9k in Pichia pastoris GS115 and subsequent integration into the genome, the vector was first linearized with Sal I and electrotransformed using a Gene Pulser Xcell electroporator:

[0063] 1) Pick a single colony of yeast, inoculate in 5mLYPD medium, and culture overnight at 30°C and 250rpm;

[0064] 2) Inoculate 100-500 μL of the overnight culture into 500 mL of LYPD medium, culture at 30°C, 250 rpm to OD 600 Reach 1.3-1.5;

[0065] 3) Centrifuge the bacterial solution at 4°C, 3000 rpm for 5 minutes, and resuspend the bacterial cells with 500 mL of ice-cold sterile water;

[0066] 4) Centrifuge according to step 3, and resuspend the bacterial cell pellet with 250 mL ice-cold sterile water;

[0067] 5) Centrifuge according to step 3, and resuspend the bacterial cell pellet with 20 mL ice-cold 1M sorbitol;

[0068] 6) Centrifuge according to step 3, and resuspend the cells ...

Embodiment 3

[0073] The screening of embodiment 3 high-copy bacterial strains and the expression of target protein

[0074] In order to obtain high-copy genetically engineered bacteria, selection pressure was used for screening. The correct single colony will be verified and seeded on the YPD plate containing different G418 concentrations with a gun tip. When spotting, spot the plates in order of G418 concentration from low to high, and culture at 30°C.

[0075] Inoculate high-copy single colonies that have passed G418 (4.0mg / mL) pressure screening in 25mL BMGY, and cultivate them to OD at 30°C and 280rpm 600 Reach 2-6. Collect cells by centrifugation at 3000g for 5min. Discard the supernatant, resuspend the cells with BMMY medium, transfer to 50mL BMMY, induce protein expression at 30°C, 280rpm; 1mL is sampled every 24h, and methanol is added once to reach a methanol concentration of 0.5%, and the induction ends after 120 hours.

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Abstract

The invention discloses an IgG antibody affinity peptide-alkaline phosphatase fusion protein which is a fusion protein formed by an IgG antibody affinity peptide and alkaline phosphatase, can be used for immunoassay as a substitute enzyme-labelled antibody. The fusion protein has the binding activity with an IgG antibody and the AP (Alkaline Phosphatase) catalytic activity once generated, the label ratio of the binding activity and the AP catalytic activity is 1:1, the enzyme specific activity of the fusion protein is 386 plus / minus 82U / mg, and an affinity constant Ka with a rabbit IgG antibody is congruent to 6.7*107L.mol-1. A preparation method of the IgG antibody affinity peptide-alkaline phosphatase fusion protein comprises the following steps of: firstly, connecting an IgG antibody affinity peptide gene with an alkaline phosphatase gene to construct a recombinant vector; secondly, carrying out electrotransformation on Pichia pastoris; thirdly, screening a high copy expression strain with G418 resistant pressure; fourthly, culturing the strain, and carrying out induced expression; and fifthly, extracting the purified IgG antibody affinity peptide-alkaline phosphatase fusion protein.

Description

technical field [0001] The invention relates to an IgG antibody affinity peptide-alkaline phosphatase fusion protein which can be used as an enzyme-labeled antibody for immunoassay, and belongs to the field of bioengineering. Background technique [0002] Alkaline Phosphatase (Alkaline Phosphatase, AP) and Horseradish Peroxidase (Horseradish Peroxidase, HRP) are more commonly used enzymes for immunolabeling in enzyme-linked immunosorbent assay (ELISA). When used in ELISA, the sensitivity of the AP system is generally higher than that of the HRP system, and the blank value is also lower. However, because AP is difficult to obtain high-purity preparations, its stability is lower than that of HRP, its price is higher than that of HRP, and the yield of enzyme conjugates is lower than that of HRP. Therefore, HRP is generally used in domestic ELISA. [0003] Alkaline phosphatase is used as a labeling enzyme in immunoassays by catalyzing the color development of the corresponding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81G01N33/535
Inventor 唐金宝杨洪鸣梁淑娟陈永
Owner WEIFANG MEDICAL UNIV
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