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A human metallothionein-1 fusion protein expression vector

A metallothionein and fusion protein technology, which is applied in the expression vector of human metallothionein-1 fusion protein, 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-1 fusion protein expression vector
  • A human metallothionein-1 fusion protein expression vector
  • A human metallothionein-1 fusion protein expression vector

Examples

Experimental program
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Effect test

Embodiment 1

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

[0027] Step 1. Artificial synthesis and optimization of hFABP-encoding DNA. In this embodiment, hFABP6 is taken as an example:

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

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

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

[0031] (4) The full...

Embodiment 2

[0048] 1. Whole gene synthesis of MT1b:

[0049] The synthetic method is a general two-step PCR method (PCR, polymerase chain reaction) to obtain MT1b encoding DNA: 1) Overlap extension PCR (Overlap PCR) uses primers as templates to synthesize full-length DNA sequences, and the 4 sequences of primers used are as follows: Shown in SEQ ID NO:31-34; 2) Using the product as a template, PCR amplifies the full-length DNA, and the sequences of the two primers used are shown in SEQ ID NO.35-36.

[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-MT1b fusion protein expression vector:

[0053] (1) The MT1b DNA sequence obtained by artificial synthesis in the previous step was subjected to Kpn I / Xho I double enzym...

Embodiment 3

[0057] Example 3 Induced expression of pET28-hFABP6-MT1b

[0058] (1) Construction of control vector pET28-MT1b

[0059] (1) PCR of MT1b

[0060] PCR primers for MT1b, upstream primer: 5'-TTTTGGTACCATGGATCCTAATTGCTCTTGTAC-3' (SEQ ID NO.35), downstream primer: 5'-TTTTCTCGAGTTACGCACAACAACGAC-3' (SEQ ID NO.36).

[0061] The PCR parameters are: 94°C / 30 seconds, 56°C / 30 seconds, 72°C / 30 seconds, 30 cycles.

[0062] (2) Restriction enzyme digestion of MT1b PCR product

[0063] The PCR product was digested with NcoI / XhoI and purified by gel electrophoresis.

[0064] 3. Treat the pET28 vector with NcoI / XhoI double enzyme digestion, electrophoresis and gel purification.

[0065] 4. T4 DNA ligase ligated the linearized pET28 vector and the insert fragment MT1b.

[0066] 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-MT1b clone.

[0067...

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Abstract

The invention discloses a human metallothionein-1 fusion protein expression vector. The up stream of an expression vector clone area contains human fatty acid binding protein (hFABP6), and the down stream of the expression vector clone area contains metallothionein-1 (MT1). The invention further discloses a method for applying the expression vector. The method for applying the expression vector comprises the basic steps of construction of an expression strain, amplification, induced expression and fusion protein purification. The hFABP6-MT1 fusion protein expression vector has the following advantages of being capable of efficiently expressing MT protein and achieving soluble expression.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, and relates to a human metallothionein-1 fusion protein expression vector, in particular to a fusion expression of human free fatty acid binding protein with chaperone-like protein function and human metallothionein. Background technique [0002] In 1957, when Margoshes and Vallee were studying the biological effects of metals, a new protein was isolated from animal organs. It contains rich sulfhydryl groups and can chelate a large amount of metal ions. This substance is called metallothionein (Metallothionein in English, Abbreviated as MT). [0003] Regarding metallothionein, from 1978 to 1999, four international symposiums were held in Switzerland, Japan, and the United States. The fifth international symposium was held in Beijing in October 2005. In 1987, my country included MT in the [863] plan, a major research project during the "Eighth Five-Year Plan" and "N...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70
CPCC07K14/825C07K2319/35C12N15/62C12N15/70
Inventor 李万波陕婧婧卢婵
Owner 孙袁野
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