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Preparation method of horse serum amyloid protein A1 and expression vector and genetic engineering bacteria thereof

A technology of amyloid protein and genetically engineered bacteria, which is applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of unstable activity, less soluble protein, and low expression level, so as to avoid mutation Refolding steps, simplified purification methods, and the effect of maintaining biological activity

Active Publication Date: 2010-12-22
DIASYS DIAGNOSTIC SYST SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, for in vitro recombinant expression and purification of SAA1, due to its protein characteristics, such as easy aggregation in vitro and low expression level, there are shortcomings such as less soluble protein obtained from expression and complicated purification methods. These shortcomings have long restricted SAA1 in vitro. Further development of research
In addition, most of the recombinant SAA1 proteins obtained by the above two methods are inclusion bodies, and 6M urea or guanidine hydrochloride must be used in the purification process to take complex methods such as denaturation and renaturation of the purified target protein, so that the obtained recombinant protein is purified biologically. Low activity and unstable activity
In addition, this purification method takes a long time, has low yield, high cost, and is not conducive to mass production

Method used

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  • Preparation method of horse serum amyloid protein A1 and expression vector and genetic engineering bacteria thereof
  • Preparation method of horse serum amyloid protein A1 and expression vector and genetic engineering bacteria thereof
  • Preparation method of horse serum amyloid protein A1 and expression vector and genetic engineering bacteria thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Cloning of the gene encoding hoSAA1 and construction of the recombinant expression plasmid pET28a(+)-hoSAA1

[0041] The steps of gene cloning and plasmid construction are as follows:

[0042] (1) The hoSAA1 gene, as shown in SEQ ID NO.1, was synthesized by Invitrogen and cloned into the pBluescriptII vector;

[0043] (2) Subcloning of the hoSAA1 gene;

[0044] ①Transform the obtained pBluescriptII-SAA1 vector into DH5a host bacteria, screen and extract the plasmid containing the target DNA fragment;

[0045] ②Then undergo double enzyme digestion and separate the target DNA fragment by agarose gel electrophoresis;

[0046] ③ After purifying the target DNA fragment with a gel recovery kit, it was directly ligated to the pET-28a(+) vector;

[0047] ④ Transform the ligated product into DH5a host bacteria, extract the plasmid, and identify it by double enzyme digestion with NdeI and HindIII. Among them, the size of the enzyme-digested fragment is consistent wi...

Embodiment 2

[0053] Large-scale expression and purification method of embodiment 2 gene recombinant bacteria

[0054] Expression of recombinant hoSAA1-polyhistidine fusion protein in Escherichia coli host

[0055] 1) Transform PET28a-hoSAA1 into Rosetta-gami Escherichia coli on an LB culture plate with kanamycin resistance (50ng / ml), culture at 37°C overnight;

[0056] 2) pick a single colony in 50ml LB medium containing 50μg / ml kanamycin, and culture overnight at 37°C and 250 rpm;

[0057] 3) 2% was inoculated into 1L LB medium (including 50ug / ml kanamycin), and the bacteria were cultivated to A600 at 37°C and 250 rpm, with an O.D value of 0.5;

[0058] 4) Add IPTG to a final concentration of 1 mM and induce culture at 37°C and 300 rpm for 3 hours;

[0059] 5) Centrifuge at 4000 rpm for 20 minutes at high speed, collect the precipitate and store it at -80°C.

[0060] Reagent for purifying recombinant hoSAA1-polyhistidine fusion protein

[0061] (1) Lysis solution: 50mM Tris-HCl pH 8.0...

Embodiment 3

[0076] Embodiment 3 Western blotting proves to recombinant protein

[0077] 1) 15% acrylamide glue, 90V, 3 hours;

[0078] 2) Electrotransfer the protein fragments on the acrylamide gel to PVDF membrane through Tris-glycine buffer solution, the conditions are 1X Tris-glycine buffer solution, 20% methanol, 70V voltage for 3 hours at room temperature;

[0079] 3) Take out the PVDF membrane after electrotransfer and wash it with 1X PBST for 30 minutes;

[0080] 4) block with PBST containing 5% skimmed milk powder for 45 minutes;

[0081] 5) Rinse the PVDF membrane with 1X PBST 3 times, 10 minutes / time;

[0082] 6) Add 8 mL of PBST containing 1:2000 diluted anti-His antibody, and react at room temperature for 1 hour;

[0083] 7) Wash the PVDF membrane three times with 1X PBST, 15 minutes / time;

[0084] 8) Add anti-mouse antibody containing 1:1000 dilution and react at room temperature for 40 minutes;

[0085] 9) Wash the PVDF membrane three times with 1X PBST, 15 minutes / time...

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Abstract

The invention provides a preparation method of soluble genetic engineering horse serum amyloid protein A1 (hoSAA1) and an expression vector and genetic engineering bacteria thereof. The expression vector provided by the invention is a plasmid vector pET28a (+) containing a gene sequence of the hoSAA1, and an hSAA1 coding sequence is arranged between restriction enzyme cutting sites of Nde I and HindIII on the pET28a (+) vector. The genetic engineering bacteria provided by the invention are Escherichia coli, which is pET28a (+)-hoSAA1 / Rosetta-gami and expressible and soluble target protein. The preparation method adopts a nickel cross-linking affinity column one-step method, the affinity column is washed twice by imidazole of different concentrations, and the method has simple and convenient operation, high yield and short consuming time and is suitable for industrialized large-scale production.

Description

technical field [0001] The present invention relates to the field of protein or polypeptide production by DNA recombination technology, more specifically, the present invention relates to the method for preparing horse serum amyloid A1 (serum amyloid A1, hoSAA1) through genetic engineering, and also relates to related expression vectors and engineering bacteria. Background technique [0002] Serum amyloid A1 (serum amyloid A, SAA1) is an acute phase response apolipoprotein, which is a group of pleomorphic proteins encoded by the same cluster of genes. Mainly synthesized by liver cells. The specific binding region of its N-terminal forms SAA1-HDL particles with high-density lipoprotein (HDL), and then regulates the metabolism of high-density lipoprotein to enhance the affinity with macrophages and transport lipids to inflammatory sites. SAA1 is an effective medium Granulocyte activator, enhances the antibacterial effect of neutrophils and inhibits fungal infection. In addit...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N15/12C07K14/47C12R1/19
Inventor 吴峻王荣芳张艳钱震斌
Owner DIASYS DIAGNOSTIC SYST SHANGHAI
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