Method for expressing soluble angiostrongylus cantonensis galectin-1 protein by using pCold carrier

Angiostrongylus, soluble technology, applied in the field of molecular cloning, can solve the problems of affecting the original biological activity of the expressed protein, affecting the expression and purification of the target protein, and there is no protein expression and purification method, so as to ensure the biological activity and reduce the protein The expression, the method is simple and the effect

Inactive Publication Date: 2015-05-06
WENZHOU MEDICAL UNIV
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Problems solved by technology

[0002] Angiostrongylus cantonensis can cause serious public health problems, how to control the central nervous system damage caused by angiostrongylus cantonensis infection, how to reduce the impact of angiostrongylus cantonensis infection and ensure adequate medical response to patients , these issues have yet to be resolved
In addition, angiostrongyliasis cantonensis often occurs in outbreaks, and the reduction of immune response in the treatment stage is the key to alleviating immunopathological reactions and improving clinical symptoms. Therefore, the current clinical regulation of immune response mainly depends on hormones, and the abuse of hormones has many disadvantages
[0003] At present, the gene sequence and protein sequence of Angiostrongylus Cantonensis galectin-1 have been recorded in GenBank, but the gelatin-1 protein of Angiostrongylus Cantonensis has not yet been expressed, and there is no such protein that is relatively complete , the specific expression and purification method, and the existing prokaryotic expression method of galectin-1 protein from other species is usually induced by Escherichia coli at 37°C. Protein expression and purification, and the protein expressed by this method is often not highly soluble, which also affects the original biological activity of the expressed protein and affects its continued use

Method used

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  • Method for expressing soluble angiostrongylus cantonensis galectin-1 protein by using pCold carrier
  • Method for expressing soluble angiostrongylus cantonensis galectin-1 protein by using pCold carrier
  • Method for expressing soluble angiostrongylus cantonensis galectin-1 protein by using pCold carrier

Examples

Experimental program
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Effect test

Embodiment 1

[0106] A method for expressing soluble Angiostrongylus galectin-1 protein using pCold vector, the steps are as follows:

[0107] (1) Primer synthesis

[0108] Primers were designed according to the cDNA sequence of the galectin-1 gene. In order to clone the target gene into the pCold III vector, the 5' end of the upstream primer was designed to add a BamH I restriction site and 6 histidine codons; the 5' end of the downstream primer was designed Add Xba I restriction site;

[0109] The primer sequences are as follows:

[0110] Upstream primer: pcold3-G-F: 5'-CGGGATCCCATCATCATCATCATCATATGTCGTCTCCTCCA-3' (sequence 1);

[0111] Downstream primer: pcold3-G-R: 5'-CTTCTAGACTACTGAATTTGAATGCCGGT-3' (sequence 2), synthesized by Shanghai Sangong Co., Ltd.;

[0112] (2) Identification of galectin-1 gene fragments

[0113] Cloning of the galectin-1 gene of Angiostrongylus Cantonensis:

[0114] The kit was used to extract the total RNA of Angiostrongylus cantonii at stage 5;

[0115]...

Embodiment 2

[0147] A method for expressing soluble Angiostrongylus Cantonensis (Angiostrongylus Cantonensis) galectin-1 protein using pCold vector, characterized in that: the steps are as follows:

[0148] (1) Primer synthesis

[0149] Primers were designed according to the cDNA sequence of the galectin-1 gene. In order to clone the target gene into the pCold III vector, the 5' end of the upstream primer was designed to add a BamH I restriction site and 6 His (histidine) codons; The 'end is designed to add an Xba I restriction site;

[0150] The primer sequences are as follows:

[0151] Upstream primer: pcold3-G-F:5'-CG GGATCC ATGTCGTCTCCTCCA-3' (SEQ ID NO: 1);

[0152] Downstream primer: pcold3-G-R:5'-CT TCTAGA CTACTGAATTTGAATGCCGGT-3' (SEQ ID NO: 2);

[0153] (2) Amplification of galectin-1 gene

[0154] Total RNA of Phase 5 of Angiostrongylus cantonensis was extracted using the TRIzol kit from Life Technologie (Cat. No.: 15596-026), and the number of worms was 20. For specific...

Embodiment 3

[0187] A method for expressing soluble Angiostrongylus Cantonensis galectin-1 protein using pCold vector, the steps are as follows:

[0188] (1) Primer synthesis

[0189] Primers were designed according to the cDNA sequence of the galectin-1 gene. In order to clone the target gene into the pCold III vector, the 5' end of the upstream primer was designed to add a BamH I restriction site and 6 histidine codon tags; the 5' end of the downstream primer was designed Design and add Xba I restriction site;

[0190] The primer sequences are as follows:

[0191] Upstream primer: pcold3-G-F:5'-CG GGATCC ATGTCGTCTCCTCCA-3' (SEQ ID NO: 1);

[0192] Downstream primer: pcold3-G-R:5'-CT TCTAGA CTACTGAATTTGAATGCCGGT-3' (sequence 2);

[0193] The 6 histidine codon tags and the galectin-1 gene are inserted between BamHI and XbaI of the original pcoldIII plasmid;

[0194] (2) Amplification of galectin-1 gene

[0195] The kit was used to extract the total RNA of Angiostrongylus cantonii...

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Abstract

The invention relates to a method for expressing a soluble angiostrongylus cantonensis galectin-1 protein by using a pCold carrier. The method comprises the following steps: (1) synthesizing a primer; (2) amplifying galectin-1 genes; (3) building galectin-1 recombinant plasmids; (4) performing inducible expression and purification on the recombinant fusion protein Galectin-1 to obtain the soluble angiostrongylus cantonensis galectin-1 protein. The method is simple and easy to operate, wide in sources of used raw materials and low in cost; an expression purification method for performing inducible expression on the soluble angiostrongylus cantonensis galectin-1 protein under low temperature is built, the expression of a escherichia coli protein is reduced, the purification efficiency of a target protein is improved, and the biology activity of the galectin-1 protein is also guaranteed. The galectin-1 protein prepared by using the method is high in solubility and lays a foundation for wide application of the galectin-1 protein.

Description

technical field [0001] The invention belongs to the technical field of molecular cloning, in particular to a method for expressing soluble Angiostrongylus galectin-1 protein by using a pCold vector. Background technique [0002] Angiostrongylus Cantonensis can cause serious public health problems. How to control the damage to the central nervous system caused by Angiostrongylus cantonensis infection, and how to reduce the impact of Angiostrongylus cantonensis to ensure an adequate medical response to patients , these issues remain to be resolved. In addition, Guangzhou Angiostrongyliasis is often outbreaks, and reducing the immune response during the treatment stage is the key to reducing the immune pathological response and improving the clinical symptoms. Therefore, the current clinical regulation of the immune response mainly relies on hormones, and the abuse of hormones has many drawbacks. [0003] At present, the gene sequence and protein sequence of Angiostrongylus Ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12C07K14/435C07K1/22
Inventor 黄慧聪李星潘赵梦静
Owner WENZHOU MEDICAL UNIV
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