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Quantitative detection method of neomycin phosphotransferase (nptii) double antibody sandwich ELISA in transgenic crops

A quantitative detection method, phosphotransferase technology, applied in the field of molecular biology and biology, can solve the problems of transgenic plants not long ago and high risk of long-term effects, and achieve high sensitivity, good repeatability and strong specificity Effect

Active Publication Date: 2015-10-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some studies now show that the kanamycin resistance gene is safe, some studies have shown that 100% positive fragments with NPT II gene sequence homology were found and confirmed in the leaf periphyll soil, while it is considered that the transgenic insect-resistant cotton The NPT II can drift to the periphyll bacteria, so it is not yet possible to draw conclusions on this
Moreover, it is not long since the world's first genetically modified plant was released on the market. Considering the long-term lag in the emergence of the risk of genetically modified products, it is still impossible to draw a final conclusion on issues such as its long-term effect and how risky it may be. , it is necessary to carry out long-term follow-up monitoring

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  • Quantitative detection method of neomycin phosphotransferase (nptii) double antibody sandwich ELISA in transgenic crops
  • Quantitative detection method of neomycin phosphotransferase (nptii) double antibody sandwich ELISA in transgenic crops

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Experimental program
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Effect test

Embodiment 1

[0035] Cloning and expression of embodiment 1, NPTII gene

[0036]According to the complete coding region sequence of the NPTII gene and the reading frame and multiple cloning site on the pET28a vector, primers (upstream primer 5'-CGCGGATCCATGAGCCATATTCAACG-3'; downstream primer: 5'-CCCAAGCTTCATATGTATCCGCTCAT-3') were designed, and the pET30a plasmid was used as Template, the amplification condition is 94°C for 5min; 36 cycles of 94°C for 30s, 54°C for 30s, 72°C for 1min; 72°C for 7min. The obtained PCR product was purified by a recovery kit, ligated with pMD18-T and sequenced. The recombinant plasmid with correct sequencing was digested by BamHI and HindIII, connected to the expression plasmid pET28a which was digested by the same double enzyme, transformed into expression competent cells BL21(DE3), induced by IPTG, and the expression of exogenous gene was measured by 12% SDS -PAGE electrophoresis analysis. After sonication, the cells were purified by High Affinity Ni-IDA R...

Embodiment 2

[0037] Embodiment 2, the preparation of NPTⅡ protein monoclonal antibody

[0038] Pure-line female BALB / c mice (4-6 weeks old) homologous to myeloma cell Sp2 / 0 were selected as immunized animals, and the immunization scheme was: 100 μg purified NPTII protein and an equal volume of Freund’s complete adjuvant for the first immunization After the emulsification was complete, the mice were subcutaneously injected into multiple points on the back; 3 and 6 weeks later, 100 μg of NPTⅡ protein was emulsified with an equal volume of Freund's incomplete adjuvant, and the mice were injected subcutaneously at multiple points on the back as the second and third immunizations. Four days before fusion, 100 μg of NPTII protein without adjuvant was injected intraperitoneally. The cells were chemically fused with PEG1500, and the positive wells on the cell culture plate were detected by indirect ELISA. After three times of subcloning, the hybridoma cell line was expanded and frozen in time to s...

Embodiment 3

[0040] Example 3, Preparation of Anti-NPTII Protein Polyclonal Antibody

[0041] Six healthy New Zealand big-eared white rabbits with a body weight of about 2 kg were selected as immunized animals, and they were observed and raised in the animal room for one week. For the first immunization, 2 mL of immunogen (1 mg / mL) was mixed with the same amount of complete Freund's adjuvant and fully emulsified, and injected into the thigh muscle; afterward, it was replaced with Freund's incomplete adjuvant, and the booster was given once every 2 weeks, and more subcutaneous doses were given on the back of the neck. Point injection. Beginning after the third immunization, about 7 days after each immunization, about 0.5 mL of blood was collected from the ear margin vein with a 1 mL sterile syringe, and the serum titer was determined by indirect ELISA. When the antiserum reached the required titer, 7-10 days after the last booster immunization, the whole blood was collected from the caroti...

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Abstract

The invention relates to a quantitative enzyme-linked immuno sorbent assay (ELISA) detection method of a neomycin phosphotransferase (NPT II) double-antibody sandwich in transgenic crops. The detection method comprises the following steps: preparing an NPT II protein as an immunogen under an in vitro condition; preparing an anti-rabbit NPT II polyclonal antibody as a coated antibody; by taking an anti-rat NPT II monoclonal antibody as a capture antibody, building and optimizing the ELISA method of the double-antibody sandwich; building a standard curve by taking preparation of a series of NPT II proteins as a standard, and verifying each index of the method. The method concretely comprises the following steps: (1) cloning and expressing an NPT II gene; (2) preparing an anti-NPT II protein monoclonal antibody; (3) preparing an anti-NPT II protein polyclonal antibody; (4) building the ELISA method of the double-antibody sandwich and the standard curve. The method is high in sensitivity, strong in specificity, simple to operate, and suitable for detection of the NPT II content in the transgenic crops, and has excellent repeatability and stability.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a double-antibody sandwich ELISA quantitative detection method for neomycin phosphotransferase (NPT II) in transgenic crops. Background technique [0002] Since the first transgenic plant came out in 1983, plant transgenic technology has brought a new revolution to agricultural production. It overcomes the limitations of traditional breeding, breaks the genetic barriers between species, accelerates the process of germplasm improvement, and achieves significant economic and social benefits. The acreage of GM crops in the world continues to expand, and the varieties of GM crops are also increasing. [0003] However, the safety of genetically modified organisms, especially the safety of genetically modified crops containing antibiotic genes has gradually become the focus of attention of governments, scientific circles and the public. Although some studies now sh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577C07K16/40C07K16/06
Inventor 柴同杰王新桐
Owner SHANDONG AGRICULTURAL UNIVERSITY
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