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Method for purifying recombinant VP1 antigen of enterovirus type 71 viruses

A purification method and enterovirus technology, applied in the field of genetic engineering recombinant protein purification, can solve the problems of low protein recovery rate, low protein expression amount, long production cycle and the like, and achieve the effects of high purity, simple operation and low cost

Active Publication Date: 2010-10-13
HANGZHOU ZHEDA ZIJIN BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The eukaryotic expression system has high protein activity due to the post-translational protein processing process that the prokaryotic expression system does not have; but it also has the disadvantages of low protein expression, long production cycle and high production cost
Although the prokaryotic expression system has high expression, short production cycle and low cost, the disadvantage is that the expression products mostly aggregate in the form of insoluble inclusion bodies, and the protein recovery rate is often very low

Method used

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  • Method for purifying recombinant VP1 antigen of enterovirus type 71 viruses
  • Method for purifying recombinant VP1 antigen of enterovirus type 71 viruses
  • Method for purifying recombinant VP1 antigen of enterovirus type 71 viruses

Examples

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example 1

[0038] Example 1. Construction and fermentation of recombinant EV71 VP1 engineering bacteria

[0039] The VP1 gene is derived from the C4 subtype of enterovirus type 71. In this example, the whole gene of VP1 was designed and synthesized with the preferred codons of Escherichia coli by using a total synthesis method. The ADI gene was double digested with Nde I and BamH I, embedded in the same digested vector pET3a to construct the vector pET3a-VP1, transformed into host cell BL21 DE3 (pLyss), and positive clones were screened and sequenced. The positive strains with correct sequencing were used as the original engineering bacteria for production. Pick a single clone and culture in 500ml LB liquid medium containing 50ug / ml ampicillin antibiotic at 37°C with shaking at 300rpm until OD 600 Reach about 1.2, inoculate into 15L fermenter (B.Braun C) until OD 600 When it reached about 20 (double distilled water was used as blank control), 1mM IPTG was added to induce expression. ...

example 2

[0040] Example 2. Renaturation

[0041] The fermentation broth was centrifuged at 6000 g for 15 minutes to collect the bacteria. The cells were broken by high-pressure homogenization at 1500 psi, and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes, and the inclusion bodies were washed twice with PBS buffer. Dissolve according to the ratio of 1g inclusion body: 15ml increasing solution, the increasing solution composition is 6M guanidine hydrochloride, 5mM EDTA, 10mM Tris, pH 8.5, stirred at 25°C for 2 hours. Slowly pour into 2000ml refolding solution (10mM PB, 100mM Arg, 10% PEG20000, pH 7.2). Stir at 15°C for renaturation for 48 hours.

example 3

[0042] Example 3. Purification

[0043] 200mL of Chelating Sepharose Fast FlowSepharose gel was loaded into the XK 50 / 30 column of GE Company, the equilibration buffer was 20mM PB, 0.5M NaCl, pH 7.0, the elution buffer was 20mM PB, 0.5M NaCl, 0.5M imidazole, pH 7.0. Equilibrate the column first with 0.2M nickel sulfate solution and then with equilibration buffer. The refolding solution was loaded onto the equilibrated column at a flow rate of 20ml / min. After the sample was loaded, the equilibrated buffer was used to wash 2 column volumes, and then eluted with a linear gradient to collect the target peak. SDS-PAGE electrophoresis was used to detect the purity of the target protein, and the Lowry method was used to detect the target protein concentration. After analysis, the amount of VP1 protein that can be obtained per gram of inclusion body is 200 mg, the protein recovery rate is 20%, and the purity is more than 95% as checked by SDS-PAGE electrophoresis.

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Abstract

The invention relates to a method for purifying a recombinant protein of the gene engineering and aims to provide a method for purifying a recombinant VP1 antigen of enterovirus type 71 viruses. The method comprises the following steps of: expressing and producing a recombinant VP1 protein of the enterovirus type 71 viruses by Escherichia coli obtain an inclusion body of the protein; dissolving the inclusion body in solubilizing liquid; and dropwise adding the solution of the inclusion body into renaturation solution under the conditions of water bath and magnetic stirring and then carrying out stirring renaturation on the mixture for 48 hours. The invention discloses the method, which can make the protein existing in a form of the inclusion body become a soluble protein by protein renaturation so as to greatly improve recovery rate of an expression product. The method adopts affinity chromatography for purification in one step. The purity is over 95 percent. The method for purifying the recombinant VP1 antigen of the enterovirus type 71 viruses has simple, convenient and time-saving operation, is favorable for large-scale industrial production, has the advantages of low cost, simple and convenient process, high purity of the purified protein and the like and can provide the diagnostic antigen for development of an EV type 71 diagnostic reagent and seroepidemiological survey.

Description

technical field [0001] The invention relates to a method for purifying genetic engineering recombinant protein. More precisely, the present invention is a method for purifying recombinant enterovirus 71 virus VP1 antigen. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus. Since the disease was first reported in 1957, it has been popular many times. In 2006, the World Health Organization listed this disease as the fourth leading cause of death. The United States, Australia, Italy, France, Holland, Spain, Romania, Brazil, Canada, Germany and other countries often have hand, foot and mouth disease caused by various types of coxsackie, echovirus and EV71. In the late 1990s, the prevalence of EV71 virus was on the rise in the Asia-Pacific region. In 1998, an EV71 epidemic broke out in Taiwan, and more than 120,000 people were infected, and 78 people died. [0003] The study found that the viruses that cause ...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/70C07K14/085C07K1/22
Inventor 吴强周林福
Owner HANGZHOU ZHEDA ZIJIN BIOTECH
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