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Recombinant protein of pulmonary prosurfactant protein-B (proSP-B) as well as preparation method and application of recombinant protein

A pulmonary surfactant, recombinant protein technology, applied in the fields of peptide/protein components, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of increasing the development and research of substances, difficult, and only occur, and achieve good broad-spectrum antibacterial. Activity, high safety, low cytotoxic effect

Inactive Publication Date: 2017-09-15
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Marnie et al. found that the antibacterial effect of mature SP-B protein only occurs in vitro. In vivo, mature SP-B and phospholipids are associated with each other. The combination of phospholipids and SP-B is relatively stable, which cannot make SP-B It acts on foreign bacteria to exert its antibacterial function, and the mature SP-B acts on the phospholipid molecular layer non-selectively, lysing the cell membrane, showing non-selective lysing of somatic cells, especially red blood cells, causing body damage such as hemolysis
Therefore, people have turned their attention to prosurfactant protein-B (proSP-B), but proSP-B is difficult to obtain by chemical synthesis due to its strong hydrophobicity, which increases the The further development and research of this kind of substance

Method used

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  • Recombinant protein of pulmonary prosurfactant protein-B (proSP-B) as well as preparation method and application of recombinant protein
  • Recombinant protein of pulmonary prosurfactant protein-B (proSP-B) as well as preparation method and application of recombinant protein
  • Recombinant protein of pulmonary prosurfactant protein-B (proSP-B) as well as preparation method and application of recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example is used to illustrate the preparation method of proSP-B recombinant protein provided by the present invention.

[0058] 1. Extraction of total RNA from rat lung tissue

[0059] (1) Total RNA was extracted from rat lung tissue using a total RNA extraction kit (purchased from GeneCopoeia, USA);

[0060] (2) Electrophoretic detection of RNA

[0061] Use a newly configured gel electrophoresis solution for electrophoresis, and a gel imager to detect and analyze the quality of RNA.

[0062] 2. Acquisition and amplification of proSP-B gene cDNA

[0063] (1) Obtain cDNA fragments

[0064] Reverse transcription reaction system:

[0065] Total RNA 1 μg

[0066] Oli(dT) 18 1μL

[0067] Free RNase Water to 13μL

[0068] React at 65°C for 10 min, then store at 4°C.

[0069] reaction system:

[0070]

[0071] Reaction conditions: react at 42°C for 60 minutes, then react at 85°C for 5 minutes, and then store at 4°C. Finally, the reverse transcription produc...

Embodiment 2

[0124] This example is used to illustrate the cytotoxicity of proSP-B recombinant protein provided by the present invention

[0125] (1) CCL-149 cells were cultured in R / MINI1640 medium containing 10% FBS.

[0126] (2) Culture stable CCL-149 cells, digest the cells with trypsin, collect the cells and dilute them with medium to adjust the cell concentration, so that there are about 5000 cells in 100 μL, inoculate in the 1st to 10th wells of a 96-well plate 100 μL of CCL-149 cells, add 100 μL of culture medium in the 11th well, 37 ° C, 5% CO 2 Cultured in a cell culture incubator for 24 hours.

[0127] (3) Add 10 μL of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 21.25 μg / mL, 15.63 μg / mL, 7.81 For μg / mL, 3.91 μg / mL purified protein obtained from step (7) in Example 1, add 10 μL of sterilized distilled water to the 10th and 11th wells.

[0128] (4) Place the 96-well plate at 37°C, 5% CO 2 Cultured in the cell culture incubator for 12h.

[0129] (5) Add 10 μL of C...

Embodiment 3

[0142] This embodiment is used to illustrate the antibacterial activity of the proSP-B recombinant protein provided by the present invention

[0143] 1. Activation of strains and preparation of bacterial suspension

[0144] (1) Cultivation of Staphylococcus aureus: activate Staphylococcus aureus on LB agar plate by streaking method, culture at 37°C for 12 hours at a constant temperature, after a single colony grows, pick a single colony and inoculate it in 10mL LB liquid medium medium, 37°C, 12h, 150r / min, cultivate, pipette gun to absorb the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and obtain 10 6 cfu bacterial suspension.

[0145] (2) Culture of Escherichia coli: activate Escherichia coli on LB agar plate by streaking method, culture at constant temperature for 12 hours at 37°C, after a single colony grows, pick a single colony and inoculate it in 10mL LB liquid medium, 37 ℃, 12h, 150r / min, cultivate...

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Abstract

The invention relates to the field of genetic engineering and discloses recombinant protein of pulmonary prosurfactant protein-B (proSP-B) as well as a preparation method and application of the recombinant protein. Specifically, the recombinant protein has the amino acid sequence shown in SEQ ID NO: 1, or an amino terminal and / or carboxyl terminal in SEQ ID NO: 1 is connected to an amino acid sequence with a tag. According to the method, the proSP-B recombinant protein is obtained by a genetic engineering method, has low cytotoxicity and shows good antibacterial activity, thereby having good practical application prospect.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a recombinant protein of pulmonary surfactant protein B propeptide, a nucleic acid encoding the recombinant protein, a recombinant plasmid, a recombinant bacterial strain, and a method for preparing pulmonary surfactant protein B propeptide Methods of recombinant proteins and their use. Background technique [0002] Since the discovery of antibiotics, they have played an important role in the treatment of human diseases, cured many infectious diseases that humans were helpless, saved many lives, and greatly increased the average life expectancy of humans. However, due to their large-scale use, abuse and irregular use, a large number of drug-resistant strains appear and spread, which complicates the treatment of infectious diseases, increases treatment costs, increases morbidity and mortality, and makes the resistance of infectious bacteria Drug property has become an u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P31/04A61P31/10
CPCA61K38/00C07K14/47C07K2319/23C12N15/70
Inventor 王万能刘钦
Owner CHONGQING UNIV OF TECH
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