Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof

A metallothionein, Huaxi crab technology, applied in the biological field, can solve the problems of difficulty in establishing a fast, accurate and standardized enzyme-linked immunoassay detection method, poor specificity of polyclonal antibodies, and uneven physical and chemical properties, etc., to improve stability. and purity, specificity, and high uniformity

Inactive Publication Date: 2013-12-25
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, polyclonal antibodies have poor specificity, limited yield, and heterogeneous physical and chemical properties, making it difficult to establish a fast, accurate, and standardized enzyme-linked immunoassay method

Method used

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  • Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof
  • Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof
  • Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Recombinant expression of metallothionein from Henan Huaxi crab.

[0035] Step 1: Selection of plasmid vector

[0036] The basic phosphate promoter (phoA) and signal sequence have several advantages in secreting and expressing foreign proteins in E. coli (see the third edition of "Molecular Cloning Experiment Guide"), which can maintain the natural conformation of foreign proteins to the greatest extent and at the same time secrete Proteins to the periplasm are more stable. In the phoA system, the phoA gene encodes the alkaline phosphatase of the phosphate scavenging system of Escherichia coli, which is inhibited in the presence of excess phosphate, and is gradually induced in the state of starvation (bacterial growth enters the lag phase). This regulation makes the accumulation of exogenous protein in the cell gradual, unlike the rapid accumulation regulated by other promoters. This chronic accumulation reduces the chances of overloading the bacterial expre...

Embodiment 2

[0045] Example 2 Preparation of anti-Huaxi crab metallothionein monoclonal antibody

[0046] Step 1: Mouse Immunization

[0047] The purified rcMT uses BSA as a standard protein, and the protein concentration is determined by the BCA method.

[0048] (1) Basic immunity:

[0049] The rcMT stored in the buffer (0.01M Tris-HCl, 0.2mM PMSF, pH7.8) was diluted to 120 μg / ml, and mixed with Quicklyantibody water-soluble adjuvant (purchased from Beijing Kangbiquan Biotechnology Co., Ltd. company) were fully mixed, and the mice were immunized by intramuscular injection, with an injection volume of 0.1ml / only. Immunization dose is 0.1ml, 20μg rcMT per mouse. After the injection, use alcohol cotton ball to disinfect the injection site to prevent infection. (2) Booster immunization: 21 days later, the mice were immunized for the second time, and the amount and method of immunization were the same as above. (3) Shock immunity: 35d tail blood collection ELISA assay. When the serum tite...

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Abstract

The invention provides a preparation method of soluble recombinant sinopotamon metallothionein (rcMT), and the preparation method has the advantages of simplicity and convenience in operation, low cost, high purity and good activity. The method comprises the following steps: firstly constructing a phoA-MT recombinant expression vector by taking a phoA plasmid as an expression vector; further transforming escherichia coli BL21, constructing an escherichia coli engineering strain phoA-MT/BL21, and inoculating the engineering strain into an LB (Luria-Bertani) culture medium to prepare seeds, then diluting bacteria to a low-phosphorus culture medium for induction and expression, and utilizing Ni sepharoseTM6 Fast Flow affinity chromatography for purification so as to get the recombinant sinopotamon metallothionein. An anti-sinopotamon metallothionein monoclonal antibody prepared by utilizing the recombinant sinopotamon metallothionein has high sensitivity and strong specificity and can be applied to ELISA (enzyme-linked immunosorbent assay) quantitative assay of the metallothionein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation of a recombinant brook crab metallothionein and a monoclonal antibody thereof. Background technique [0002] Metallothionein (MT) is a class of widely distributed, small molecular weight (6000-7000Da), rich in cysteine, free of aromatic amino acids and histidine, which can be induced by metals and bind multiple metal atoms. Special non-enzymatic proteins. In 1957, Margoshes and Vallee of Harvard University isolated and named horse kidney for the first time. Subsequent studies found that MT widely exists in animals, plants and microorganisms. Studies have shown that MT is involved in the storage, transport and metabolism of trace elements in the body, has the functions of antagonizing ionizing radiation, scavenging free radicals and detoxifying heavy metals, and is also related to the growth and development of the body, delaying aging and certain diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K16/18
Inventor 何永吉王兰马文丽刘进平井维鑫
Owner SHANXI UNIV
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