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Human metallothionein-4 fusion protein expression vector

A metallothionein and expression vector technology, applied in the fusion expression of human free fatty acid binding protein and human metallothionein, and the field of human metallothionein-4 fusion protein expression vector, can solve the problems of high cost and low yield.

Active Publication Date: 2016-03-30
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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  • Human metallothionein-4 fusion protein expression vector
  • Human metallothionein-4 fusion protein expression vector
  • Human metallothionein-4 fusion protein expression vector

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Experimental program
Comparison scheme
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, and use the translated protein The amino acid sequence homology 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 multiple cloning region (SEQ ID NO. 3).

[0031] (4)...

Embodiment 2

[0048] 1. Whole gene synthesis of MT4:

[0049]The synthetic method is a general two-step PCR method (PCR, polymerase chain reaction), and obtains MT4 coding DNA: 1) Overlap extension PCR (OverlapPCR) uses primers as templates to synthesize full-length DNA sequences, and 4 primers (SEQ ID NO: 23-26); 2) using the product as a template to amplify the full-length DNA by PCR, 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-MT4 fusion protein expression vector:

[0053] (1) The MT4 DNA sequence obtained by artificial synthesis was purified by KpnI / XhoI double enzyme digestion, electrophoresis, and gel cutting;

[0054] (2) At the same time, the pET28-hFABP6 vector was ...

Embodiment 3

[0058] (1) Construction of the control vector pET28-MT4

[0059] PCR primers for MT4, upstream primer: 5'-TTTTGGTACCATGGATCCGCGCGAATGTGTCTGCATGTCTG-3' (SEQ ID NO.27), downstream primer: 5'-TTTTCTCGAGTTATGGACAGCAGGAGCATTTATC-3' (SEQ ID NO.28).

[0060] The PCR parameters are: 94°C / 30 seconds, 56°C / 30 seconds, 72°C / 30 seconds, 30 cycles.

[0061] (2) Restriction enzyme digestion of MT4 PCR product

[0062] The PCR product was digested with NcoI / XhoI and purified by gel electrophoresis.

[0063] 3. Treat the pET28 vector with NcoI / XhoI double enzyme digestion, electrophoresis and gel purification.

[0064] 4. T4 DNA ligase ligated the linearized pET28 vector and the insert fragment MT4.

[0065] 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-MT4 clone.

[0066] 6. Take the above-mentioned correct clones and culture them in LB medi...

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Abstract

The invention discloses a human metallothionein-4 fusion protein expression vector. The upstream of an expression vector cloning region comprises human heart-type fatty acid-binding protein (hFABP6), and the downstream comprises the human metallothionein-4 (MT-4). The invention also reveals an application method of the expression vector, and the method comprises the following basic steps: construction of an expression bacterial strain, amplification, inducible expression and purification of fusion proteins. The hFABP6-MT4 fusion protein expression vector has the following characteristics (1) high-efficiency expression of MT4 proteins; (2) soluble expression.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, and relates to a human metallothionein-4 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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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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