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Method for preparing enterotoxin C2 protein

A technology of enterotoxin and protein, which is applied in the field of preparation of enterotoxin C2 protein, can solve the problems of limited output, long time consumption, cost limitation of excision efficiency and wide use of protease, and achieve the effect of simple and easy operation

Inactive Publication Date: 2009-06-17
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are mainly two methods for preparing SEC2. One is to obtain the natural wild-type SEC2 protein from the culture medium of Staphylococcus aureus secreting wild-type SEC2 by means of ion exchange and gel exclusion. However, the amount of SEC2 secreted by Staphylococcus aureus itself is very small, and the content in the culture medium is extremely low (nanogram level), and the pure product can only be obtained through three column chromatography. The operation process is complicated, time-consuming, and the protein loss is huge. , the output is severely limited
Another way is to express a large amount of recombinant SEC2 protein with a purification tag (such as histidine tag His-tag or glutathione transferase tag GST) in Escherichia coli in a genetic engineering manner, and use affinity chromatography ( Such as Ni 2+ Ion affinity chromatography or glutathione affinity chromatography), this method has a large yield and less time-consuming, but the introduced purification tags are all bound to the recombinant SEC2 molecule in the form of fusion protein, affecting its spatial conformation affect its biological activity
Although there are commercially available proteases that can cleave the fused purification tag, their excision efficiency, repurification process after excision, and the cost of the protease itself limit their widespread use.

Method used

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  • Method for preparing enterotoxin C2 protein
  • Method for preparing enterotoxin C2 protein
  • Method for preparing enterotoxin C2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Extraction of template DNA: Inoculate a single colony of Staphylococcus aureus producing wild-type SEC2 into 5 ml of liquid LB medium, culture overnight on a shaker at 37° C., and collect 1.5 ml of the culture by centrifugation to collect the bacteria. Extract Staphylococcus aureus genomic DNA as PCR reaction template (genome DNA extraction operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Stern Lal's "Refined Molecular Biology Experiment Guide", New York John Wiley & Sons Publishing House, 1995, third edition, P39-40).

[0028] (2) PCR amplification:

[0029] Use primers with NcoI and XhoI restriction sites and 5' end protection bases respectively:

[0030] wsec-F: 5'-CG CCATGG AGAGTCAACCAGA-3', wsec-R:5'-TCG CTCGAGTTATCCATTCTTTGTTG-3' (the underlined parts are Nco I and Xho I restriction sites respectively, and the italic font is the 5' end protection base) PCR reaction system is: 10×Pyrobest reaction buffer 5 μ...

Embodiment 2

[0034] Construction of prokaryotic heterologous expression vector pet28a-sec2:

[0035] The purified PCR product fragments were digested with NcoI and XhoI restriction endonucleases, and then ligated into the pET-28a expression vector that had been cut by the same double enzymes with T4 DNA ligase to construct an expression vector pet28a-sec2, which was transformed into E.coli BL21( DE3) Competent cells (transformation operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Stellar "Refined Molecular Biology "Experiment Guide" New York John Wiley & Sons Publishing House, 1995 Third Edition P39-40), extract plasmid DNA, identify correct recombinant clones by EcoRI, XhoI double enzyme digestion, and determine DNA sequence by Sanger dideoxy terminal termination method.

[0036] The detected DNA fragment has the amino acid sequence in the sequence table SEQ ID NO: 1:

[0037] ESQPDPTPDE LHKSSEFTGT MGNMKYLYDD HYVSATKVMS VDKFLAHDLI 50

[...

Embodiment 3

[0054] Heterologous expression of protein in E. coli BL21(DE3).

[0055] (1) Inoculate a single colony of BL21(DE3) transformed with the pET-28a-sec2 plasmid in liquid LB medium containing 40ug / ml kanamycin, activate overnight culture, and transfer to the next stage with 1% volume inoculum , cultured on a shaker at 37°C until OD 600 0.6, add IPTG at a final concentration of 1.0 mmol / L, and induce expression at 30°C for 6 hours. Centrifuge at 4000rmp for 10 minutes to collect the thalline, take a small amount of sample with 2× loading buffer ((loading buffer is 100 mg sodium dodecylsulfonate (SDS), 2 g glycerol, 2 mg bromophenol blue, 0.1 ml β-mercaptoethanol , and with 50mM Tris-HCl (pH8.0) fixed volume to 10ml) processing, processing method according to Wang Jiazheng, Fan Ming "Protein Technology Handbook" Science Press 2002 first edition P81 and P87), 12% SDS-PAGE electrophoresis (electrophoresis conditions: 50 volts, 30 minutes, then 120 volts, 2 hours), Coomassie brillia...

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Abstract

The invention relates to a genetic engineering technique, especially to a method for preparing an enterotoxin C2 protein comprising the following steps: (1) amplifying a gene fragment which codes the golden yellow staphylococcus enterotoxin according to the conventional PCR by using the golden yellow staphylococcus genome as templates and enzyme sites with Nco I and Xho I and 5' terminal protective bases as primers, and connecting obtained an objective gene to a vector; (2) converting plasmids constructed by a subcloning technology to the bacillus coli, and inducing protein expression by using conventional method IPTG; (3) purifying the staphylococcus aureus enterotoxin C2 protein by using an affinity purification column combining metal ions. The enterotoxin C2 protein of the invention does not contain any induced excessive amino-acid residue, thereby remaining a completely biology activity as a wild-type SEC2 expressed by the golden yellow staphylococcus. Moreover the purity is more than 98 The method of the invention is simple and is easy to operate, and the purification speed and purity are obviously high.

Description

technical field [0001] The invention relates to genetic engineering technology, in particular to a preparation method of enterotoxin C2 protein. Background technique [0002] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which can stimulate the proliferation of most T cells at a very low concentration (1-10 ng / mL), and has a super-strong function of enhancing the body's immune response and certain anti-inflammatory effects. tumor activity. Therefore, superantigen is an excellent immunomodulator and synergist, and it is expected to be developed into a potential new antitumor drug for tumor treatment. [0003] Staphylococcal enterotoxins (SEs) are a representative class of microbial exotoxins. Because of its extremely strong T cell activation function, it has become a typical microbial superantigen and has been widely valued by people. In recent years, people have done a lot of research work on the structure, immunological function and a...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/31C12N15/70C07K14/195
Inventor 徐明恺张惠文张成刚王小刚
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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