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A human metallothionein-2a fusion protein expression vector

A metallothionein and expression vector technology, applied in the field of human metallothionein-2a fusion protein expression vector, fusion expression of human free fatty acid binding protein and human metallothionein, can solve the problems of high cost and low yield.

Active Publication Date: 2018-11-23
孙袁野
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, MT products are mainly extracted from rabbit liver, horse kidney, pig liver and microorganisms (such as Neurospora crudes), etc. The high cost and low yield inhibit her from being widely used, such as food, beverages, cosmetics, etc.

Method used

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  • A human metallothionein-2a fusion protein expression vector
  • A human metallothionein-2a fusion protein expression vector
  • A human metallothionein-2a fusion protein expression vector

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Experimental program
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Effect test

Embodiment 1

[0026] A construction method for a fusion protein expression vector of a chaperone-like protein, comprising the following steps:

[0027] Step 1, artificial synthesis and optimization of the coding DNA of hFABP, this embodiment takes hFABP6 as an example:

[0028] (1) Take the hFBP6 amino acid sequence (SEQ ID NO.2) and reversely translate it into a DNA coding sequence, and obtain the sequence (SEQ ID NO.1) through codon preference and ribosome binding region sequence optimization, but not limited to this sequence, with The amino acid sequence homology of the translated protein is limited to be equal to or greater than 85%.

[0029] (2) The coding sequence was segmented to synthesize oligonucleotide single-stranded DNA (SEQ ID NO. 5-18).

[0030] (3) Synthesis of full-length coding DNA sequence by polymerase chain reaction (PCR) method, including 5'-6xHisTag (SEQ ID NO. 4), hFBP6 amino acid coding sequence, 3'-flexible linker sequence (Linker) and polyclonal region (SEQ ID NO...

Embodiment 2

[0048] 1. Whole gene synthesis of MT2a:

[0049]The synthesis method is a common two-step PCR method (PCR, polymerase chain reaction) to obtain MT2a coding DNA. 1) Overlap PCR (Overlap PCR), using primers as templates to synthesize a full-length DNA sequence, 4 primers (SEQ ID NO: 23-26) were used. 2) Using the product as a template, PCR amplifies the full-length DNA, using 2 primers (SEQ ID NO.27, 28).

[0050] (1) Head extension PCR parameters: 94°C / 30 seconds, 56°C / 30 seconds, 72°C / 30 seconds, 15 cycles, complement extension 72°C / 2 minutes.

[0051] (2) Full-length synthetic PCR parameters: 94°C / 30 seconds, 58°C / 30 seconds, 72°C / 45 seconds, 30 cycles, complement extension 72°C / 5 minutes.

[0052] 2. Construction of hFABP6-MT2a fusion protein expression vector:

[0053] (1) The MT2a DNA sequence obtained by artificial synthesis in the previous step was subjected to Kpn I / Xho I double enzyme digestion treatment and purification;

[0054] (2) At the same time, the pET28-hF...

Embodiment 3

[0058] (1) Construction of control vector pET28-MT2a

[0059] (1) PCR of MT2a

[0060] PCR primer of MT2a, upstream primer: 5'-TTTTGGTACCATGGATCCTAATTGCTCTTGTAC-3' (SEQ ID NO.27),

[0061] Downstream primer: 5'-TTTTCTCGAGTTACGCACAACAACGAC-3' (SEQ ID NO.28).

[0062] The PCR parameters are: 94°C / 30 seconds, 56°C / 30 seconds, 72°C / 30 seconds, 30 cycles.

[0063] (2) Restriction enzyme digestion of MT2a PCR product

[0064] The PCR product was digested with NcoI / XhoI and purified by gel electrophoresis.

[0065] 3. Treat the pET28 vector with NcoI / XhoI double enzyme digestion, electrophoresis and gel purification.

[0066] 4. T4 DNA ligase ligated the linearized pET28 vector and the insert fragment MT1b.

[0067] 5. Transform the ligation product into Escherichia coli competent cell BL21(DE3), pick a single colony, amplify and identify the recombinant, and sequence and analyze to obtain the correct pET28-MT2a clone.

[0068] 6. Take the above-mentioned correct clones and cul...

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Abstract

The invention discloses a human metallothionein-2a fused protein expression vector. The upstream of the expression vector contains human fatty acid binding protein (hFABP6), and the downstream is metallothionein (MT2a). The invention also discloses an application method of the expression vector. The method comprises the basic methods of expression strain building, amplification, inducible expression and fusion protein purification. The hFABP6-MT2a fused protein expression vector disclosed by the invention has the following characteristics that 1, the MT2a protein is efficiently expressed; 2, the dissolubility expression is realized.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and fermentation engineering, and relates to a human metallothionein-2a fusion protein expression vector, in particular to the fusion expression of a human free fatty acid binding protein with a chaperone-like protein function and a human metallothionein. Background technique [0002] In 1957, when Margoshes and Vallee were studying the biological role of metals, they isolated a new protein from animal organs. It is rich in sulfhydryl groups and can chelate a large number of metal ions. referred to as MT). [0003] Regarding metallothionein, from 1978 to 1999, four international symposiums were held successively in Switzerland, Japan and the United States. The fifth international symposium was held in Beijing in October 2005. In 1987, our country included MT in the [863] plan, the "Eighth Five-Year Plan" and "Ninth Five-Year Plan" major key research projects, and in 1994 it was included in the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N15/62
CPCC07K14/47C07K14/825C07K2319/21C12N15/63C12N15/70
Inventor 李万波陕婧婧卢婵
Owner 孙袁野
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