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Human thyroid stimulating hormone receptor fusion protein as well as preparation method and application thereof

A technology of thyroid stimulating hormone and fusion protein, applied in the biological field, can solve problems such as low expression and stability

Active Publication Date: 2020-08-18
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion protein solves the problem of low expression and stability, making the expression as high as 100mg / L

Method used

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  • Human thyroid stimulating hormone receptor fusion protein as well as preparation method and application thereof
  • Human thyroid stimulating hormone receptor fusion protein as well as preparation method and application thereof
  • Human thyroid stimulating hormone receptor fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Using CHO cells to construct a system for transiently expressing fusion proteins

[0031] (1) Construction of a plasmid containing the target gene

[0032] According to the experimental requirements, the PCMV3 plasmid was selected as the backbone, and the TSHR target gene fragment was inserted into the multiple cloning site. The PCR systems in Table 1 and Table 2 and the PCR program in Table 3 were used to amplify the gene fragments of HER2 and TSHR, respectively.

[0033] Table 1 PCR system

[0034]

[0035]

[0036] Table 2 PCR system

[0037]

[0038] Table 3 PCR program

[0039]

[0040] Glue recovery:

[0041] The DNA fragments were run on 1% DNA agar gel, and then recovered with a recovery kit, and then splice PCR was performed.

[0042] Splicing PCR:

[0043] The primer information is as follows. After PCR, the gel is recovered, and then digested.

[0044] Table 4 PCR system

[0045]

[0046]

[0047] Table 5 enzyme digestion s...

Embodiment 2

[0137] Example 2 Constructing a system for stably expressing fusion proteins with CHO cells

[0138] (1) Construction of PMKO-TSHR-HER2 plasmid

[0139] The TSHR-HER2 fragment was inserted into the PMKO plasmid from the plasmid, and the method was as described in Experiment 1 above.

[0140] (2) Preparation of virus

[0141] The constructed plasmids and viral packaging plasmids VSVG and GAG were transfected into CHO cells with PEI. After 12 hours of transfection, the cell culture medium was removed, washed twice with PBS, and replaced with fresh cell culture medium. Collect the virus 36 hours after changing the medium, store it at 4°C within a week, and store it at -80°C for a long time.

[0142] (3) Virus infects target cells

[0143] Add 4 ml of virus to a 10 cm dish, add 2 ml of virus to a 6 cm dish, add 1 ml of virus to a 3.5 cm dish, and add polybrene to increase the efficiency of virus infection. After 5 hours of infection, add fresh cell culture medium, add 4 ml of...

Embodiment 3

[0148] Embodiment 3 purifies fusion protein

[0149] The purification method of the fusion protein utilizes the principle of his column metal chelation chromatography. The operation method is as follows:

[0150] Buffer:

[0151] Loading buffer: 0.02M phosphate, 0.5M NaCl 5-50mM imidazole pH7.4

[0152] Elution buffer: 0.02M phosphate, 0.5M sodium chloride, 0.5M imidazole pH7.4.

[0153] 1) Collect the cell supernatant, and then take a sample for SDS-PAGE electrophoresis analysis.

[0154] 2) Concentrate the supernatant in a hollow fiber column, and then take a sample for SDS-PAGE electrophoresis analysis.

[0155] 3) Chromatographic column preparation: use 0.1M nickel sulfate (NiSO 4 ) to fill the column with Ni 2+ , and then flush at least 5 column volumes with ultrapure water.

[0156] 4) Sample purification steps:

[0157] a. Equilibrate 5-10 column volumes with loading buffer.

[0158] b. Sample loading.

[0159] c. Wash 5-10 column volumes with loading buffer. ...

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Abstract

The invention relates to the technical field of biology, in particular to a human thyroid stimulating hormone receptor fusion protein and a preparation method and application thereof. The fusion protein is composed of a HER2 protein and a TSHR protein, and the HER2 protein and the TSHR protein are connected through a connecting peptide. The HER2 and the low-expression TSHR are subjected to fusionexpression, the yield and stability of the TSHR can be improved under the condition that the structure and function of the TSHR are not affected, the TSHR protein can be secreted into supernate, and purification is facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human thyroid-stimulating hormone receptor fusion protein and its preparation method and application. Background technique [0002] Human thyroid-stimulating hormone receptor (TSHR) mainly exists on the membrane of thyroid follicle cells, and partly distributes in hepatocytes, lymphocytes, adipocytes, retroocular fibroblasts, neurons and astrocytes. hTSHR is a seven-transmembrane protein, which belongs to the subfamily of G protein-coupled receptors, glycoprotein receptors. The protein has a large extracellular region, a transmembrane region and an intracellular region. The extracellular region is responsible for binding protein ligands, including glycoprotein hormones and relaxin, and its transmembrane region is responsible for receptor activation and signal transduction of G protein, activates adenylate cyclase to generate cAMP, and initiates the second signal of cell cAMP The s...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/02C12N15/70G01N33/68
CPCC07K14/723C12N9/0071C12Y114/18001C12N15/70G01N33/6854C07K2319/00
Inventor 田晓平刘彩霞赵巧辉李桂林付光宇吴学炜杨增利
Owner ZHENGZHOU IMMUNO BIOTECH
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