Cloning of chicken CR2 gene, expression and purification of protein as well as preparation of polyclonal antibody of protein

A cloning vector, expression vector technology, applied in the field of molecular cloning, to achieve the effect of enhanced prevention and control

Inactive Publication Date: 2019-11-08
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in birds, the CR2 gene is rarely reported, especially the CR2 gene of poultry chicken has not been annotated

Method used

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  • Cloning of chicken CR2 gene, expression and purification of protein as well as preparation of polyclonal antibody of protein
  • Cloning of chicken CR2 gene, expression and purification of protein as well as preparation of polyclonal antibody of protein
  • Cloning of chicken CR2 gene, expression and purification of protein as well as preparation of polyclonal antibody of protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Amplification of ChCR2 gene, and construction of plasmids with different tags

[0037] Using RNASeq experimental technology to measure chicken spleen lymphocytes, several new chicken genes that have not been annotated were obtained. Through gene comparison, it was found that one of the new genes had more than 80% similarity with CR2 of other birds, and it was found through structure prediction The amino acid sequence of the new gene has a CR2 domain; according to the RNASeq sequencing results, the sequenced new gene was compared with the GenBank database, and one of the new genes (ENSGALG00000030007) was found to be the same as the Gallus gallus mRNA for hypothetical protein, clone 31a4 registered in GenBank (GenBank: AJ720954.1) sequence results are consistent, the gene has a CR2 domain, design specific primers ChCR2-F and ChCR2-R according to the gene sequence, send to the company for synthesis, and amplify the ChCR2 gene fragment by RT-PCR. Specifically:...

Embodiment 3

[0057] Example 3 protein purification experiment

[0058] Purification of GST-ChCR2-ΔTM soluble protein: Collect Transetta competent cells expressing GST-ChCR2-ΔTM, ultrasonically lyse and take the supernatant, and add the supernatant to the processed Glutathione Sepharose TM 4 Incubate in the Fast Flow resin filler at 4°C for 6 hours, wash the filler with 10 times the column volume of PBS, and finally use 15ml of reduced glutathione eluent to elute the protein bound to the filler. Run SDS-PAGE to analyze the purity of the purified protein, and finally get a relatively pure GST-ChCR2-ΔTM protein, the results are as follows Figure 4 As shown, 1. supernatant, 2. flow-through, 3. purified GST-CR2-ΔTM protein.

[0059] Purification of His-ChCR2-ΔTM inclusion body protein: collect Transetta competent cells expressing His-ChCR2-ΔTM, sonicate to obtain the precipitate, and use a glass rod to remove the yellow cell debris on the upper layer of the inclusion body precipitate, leavin...

Embodiment 4

[0060] Example 4 Preparation of ChCR2-ΔTM polyclonal antibody

[0061] 1. Immunization of Balb / c mice.

[0062] Six 8-week-old healthy female BABL / c mice were immunized every two weeks according to the immunization program shown in Table 1.

[0063] Table 1 Immunization program

[0064]

[0065] 2. Determination of serum titer by ELISA

[0066] One week after the fourth immunization, eyeball blood was collected, serum was collected, and serum titer was determined by indirect ELISA method. The purified His-ChCR2-ΔTM protein was used as the coating antigen, the coating concentration of the antigen was 16 μg / mL, and the coating condition was 37°C for 2h+4°C overnight, and the serum titer of the mice was determined. The results showed that compared with the negative control, the serum titers of the 6 mice could all reach 1:512000, and the results were as follows: Image 6 shown.

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Abstract

The invention provides cloning of chicken CR2 gene, expression and purification of protein as well as preparation of a polyclonal antibody of protein. An amino acid sequence and a gene sequence of ChCR2 protein are provided, a ChCR2 prokaryotic expression vector containing different labels is constructed, an Escherichia coli prokaryotic expression system is used for expressing ChCR2 protein, and the protein is purified. The purified protein can prepare the polyclonal antibody with higher titer. The ChCR2 gene plays important roles in a chicken immune system. CR2 can link congenital complementmedicated immune response with pathogens and foreign antigens and combine with C3d covalently attached to a target to produce adaptive immune response, and caused cell signaling phenomenon can greatlyreduce threshold of B cell activation. B cells are important in the immune system, so that the invention finds that ChCR2 is of great significance in both fundamental research and clinical application.

Description

technical field [0001] The invention belongs to the technical field of molecular cloning, and in particular relates to the cloning of a new chicken gene CR2 (Chicken Complement Receptor 2, ChCR2), the expression and purification of the protein and the preparation of polyclonal antibodies. Background technique [0002] Complement receptor 2 (CR2, complement receptor 2), CD21, is the receptor of complement C3 cleavage fragments C3d, C3dg, etc., and is a glycoprotein with a transmembrane region on the surface of B cells, mainly expressed in mature B cells and filter surface of dendritic cells. CR2 was also found on human CD4+ and CD8+ T cell subsets, astrocytes and pharyngeal epithelial cells. CR2 is expressed on mature B cells, but not on pre-B or immature B cells, nor is it found when activated B cells differentiate into plasma cells. CR2 is a member of the C3 / C4 binding protein structural family and is encoded by regulators of the complement activation (RCA, regulators of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K16/28C07K16/06C07K1/16C12N15/12C12N15/70C12N15/66
CPCC07K14/70596C07K16/2896C07K16/065C12N15/70C12N15/66
Inventor 李永清靳换孔子萌江波许健刘文晓
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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