Gene engineering preparation method of bioactive peptide containing human alpha defensin 5

A genetic engineering and defensin technology, applied in the field of bioengineering pharmacy, can solve problems such as the preparation process that has not yet been established

Inactive Publication Date: 2009-09-09
ARMY MEDICAL UNIV
View PDF1 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Several foreign pharmaceutical companies are conducting research and development on defensins, but the existing published literature shows that defensins molecules are

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Gene engineering preparation method of bioactive peptide containing human alpha defensin 5
  • Gene engineering preparation method of bioactive peptide containing human alpha defensin 5
  • Gene engineering preparation method of bioactive peptide containing human alpha defensin 5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Cloning of human α-defensin 5 gene (HD5)

[0114] Obtain the cDNA sequence of HD5 gene by RT-PCR method from the ileal mucosa sample of patients with common enteritis (the sample in this example is from the First Affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army), and the PCR upstream and downstream primers used for amplification are respectively HD -sense01 and HD-antisense01. The base sequences of the primers are as follows (SEQ ID NO: 3-4 in sequence):

[0115] HD-sense01: 5'-CGT AAG CTT ATA TCC ACT CCT GCT CTC CCT-3'

[0116] HD-antisense01: 5'-ATC GCG GCC GCA ATG CTT GAA CTT TAT TTT G-3'

[0117] Refer to the instructions of the RT-PCR kit (One Step RNA PCR Kit) to establish the RT-PCR reaction system as follows: 5.0 μl 10 mM dNTP, 10.0 μl 25 mM MgCl 2 , 1.0μl RNase Inhibitor (40U / μl), 2.0μl each primer (20mmol / L), 2.0μl RNA, 1.0μl AMV RtaseXL (5U / μl), 1.0μl Taq enzyme, 5.0μl 10× buffer, 21.0μl ddH ...

Embodiment 2

[0117] Refer to the instructions of the RT-PCR kit (One Step RNA PCR Kit) to establish the RT-PCR reaction system as follows: 5.0 μl 10 mM dNTP, 10.0 μl 25 mM MgCl 2 , 1.0μl RNase Inhibitor (40U / μl), 2.0μl each primer (20mmol / L), 2.0μl RNA, 1.0μl AMV RtaseXL (5U / μl), 1.0μl Taq enzyme, 5.0μl 10× buffer, 21.0μl ddH 2O. Then follow the following procedures: ①reverse transcription: 50°C, 30 minutes; ②denaturation: 94°C, 2 minutes; ③denaturation: 94°C, 30 seconds; ④refolding: 55°C, 30 seconds; ⑤extension: 72°C , 45 seconds; ⑥Return to step "③", 34 cycles; ⑦Extend: 72°C, 7 minutes; ⑧Save at 4°C, the total number of cycles is 32 times. The obtained RT-PCR product was subjected to 1% agarose gel electrophoresis (80V, electrophoresis for 30 minutes), and the result showed that a DNA band with a size of about 450bp was amplified. After the PCR product was purified and recovered, it was ligated with the pMD18-T vector. The ligation product was transformed into DH5α Escherichia coli co...

Embodiment 3

[0139] Example 3 Fermentation of Yeast Engineering Strain and Purification of Recombinant Human α-Defensin 5 Active Peptide

[0140] Inoculate pPIC9K-mHD5-integrated yeast engineered strains frozen at -70°C on YPD plates for overnight culture and activation at 30°C, select a single colony with a good appearance and inoculate it in BMGY medium, and culture in a constant temperature shaker at 28°C and 30°C at 220rpm for 16 ~18h to OD 600 ≈3~5, then transfer to OD once at 1:10 600 It is about 4, and the bacterial liquid is used as the seed liquid. Then the seed solution is transplanted into the basal salt medium (adding trace salt PTM 1 ) in a 15L fermenter (B.Braun company, Germany) for high-density culture and fermentation. The fermentation temperature was controlled at 30°C (28°C after methanol induction), the dissolved oxygen content was controlled between 30% and 40%, the pH value was controlled at 5.0 (3.5 after methanol induction), and the cells were cultured to OD 6...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a gene engineering preparation method of bioactive peptide containing human alpha defensin 5 and discloses a method for constructing expression vector of bioactive peptide gene containing human alpha-defensin 5 and for screening and fermenting in high density transformed yeast cloning strain thereof and the method for separating and purifying the bioactive peptide in fermentation supernatant. The invention also discloses the application of the bioactive peptide containing human alpha defensin 5 in preparing polypeptide medicine. The invention is applicable to realization of high efficiency fermentation expression through simple operation in the industrialized production scale and low-cost and high-efficiency purification and preparation of the bioactive peptide containing human alpha defensin 5 capable of highly effectively killing multiple pathogenetic bacteria and resisting virus such as HPV.

Description

technical field [0001] The invention belongs to the field of bioengineering pharmacy, and in particular relates to a genetic engineering preparation method of a human α-defensin 5 active peptide with bacteria-killing and antiviral bioactive polypeptides. Background technique [0002] Bacterial and viral infections are still a major threat to human life and health. Since the advent of penicillin and sulfa drugs in the early last century, humans have invented nearly a hundred kinds of antibiotics that mainly interfere with bacterial nucleic acid or protein metabolism and synthesis or inhibit bacterial cell wall synthesis to achieve antibacterial purposes. However, due to these traditional antibiotics The mechanism of action can easily induce bacteria to mutate and produce drug resistance. A variety of drug-resistant strains against traditional antibiotics have been found in clinical practice, making humans face the challenge of pathogenic bacteria again. Therefore, people ha...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/81C12P21/02C12R1/84
Inventor 王艾平王军平粟永萍程天民王崧申明强陈默
Owner ARMY MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap