Polypeptide with Beta-lactamase inhibiting activity and its coded DNA

A technology of lactamase and DNA sequence, which is applied in the direction of recombinant DNA technology, medical preparations containing active ingredients, hydrolytic enzymes, etc., can solve the problems of drugs that have not been expressed by genetic engineering, and achieve easy cloning and expression, and improved expression The effect of small amount and molecular weight

Inactive Publication Date: 2006-05-31
SHANGHAI INST OF PHARMA IND
View PDF0 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Judging from foreign literature reports, the current research is still a relatively large β-lactamase-binding protein. Due to its relatively large molecular weight, consisting of 165 amino acids, it is not suitable for development as a drug for clinical use, even if there is research on the expression of BLIP (See: Liu HB, et al.JBiotechnol.2004 Mar 18; 108(3): 207-17.),

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
  • Polypeptide with Beta-lactamase inhibiting activity and its coded DNA
  • Polypeptide with Beta-lactamase inhibiting activity and its coded DNA
  • Polypeptide with Beta-lactamase inhibiting activity and its coded DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Expression and purification of β-lactamase

[0061] The pYG111 plasmid contains the TEM-1 type β-lactamase gene encoded in the pBR322 plasmid, and EcoRI and BamHI restriction sites were added on both sides of the gene when it was cloned. In order to express a large amount of TEM-1 type β-lactamase gene, the plasmids pYG111 and pLY-5 were digested with EcoRI and BamHI respectively, and the small fragment of β-lactamase gene excised from pYG111 and the large fragment of pLY-5 vector were recovered respectively , were ligated with T4 ligase to obtain the high-expression plasmid pYG201 of β-lactamase, and the construction process is shown in Figure 1.

[0062] After the induced cell culture solution was centrifuged at 8000r / min for 10min, the bacterial cells were collected, and the precipitated bacterial cells were washed once with distilled water, and centrifuged again. The obtained cells (wet weight 0.25 g) were washed once with 1×PBS and then suspended, and the cells we...

Embodiment 2

[0067] Construction of Oligonucleotide Libraries

[0068] Using the PCR method to amplify single-stranded random oligonucleotides into double-stranded ones is different from ordinary PCR amplification reactions. The principle of random oligonucleotide fragment amplification is shown in Figure 6. The template in this PCR reaction is a random sequence containing 9 NNYs, which contains a very large amount of information. Try to keep the number of oligonucleotides constant during PCR amplification to ensure the diversity of random sequence DNA when constructing oligonucleotide libraries.

[0069] Connect the PCR digested product with an appropriate amount of pGAD424 large fragment cut with the same enzyme, and transform E.coli competent cells (1000ml culture medium) according to the above-mentioned plasmid DNA amount enlarged 100 times, spread 100 LB+Amp plates, place in Cultivate in a 37°C incubator for 16 hours, and each plate will be covered with colonies up to 10 5 clone. C...

Embodiment 3

[0071] Screening of yeast two-hybrid system

[0072] Use 100 μg of pYG202 and pYG111 to co-transform Y153 competent cells prepared with 1 L of culture medium, spread 100 SD-L-W-H+3-AT (25mM) plates, pick out the larger colonies, and pick them together 6000 spots were planted on SD-L-W-H+3-AT (25mM) plates. When the colony grows to an appropriate size, screen and detect the color reaction of β-galactosidase activity. After preliminary screening, 62 positive transformants were obtained, and the blue colonies that appeared on the nitrocellulose filter were found on the original plate to find the corresponding colonies.

[0073] Firstly, the plasmid DNA in the positive transformants obtained by the above-mentioned preliminary screening was separated by enzymatic method, but here the isolated plasmid DNA was a mixture of two kinds of plasmids, and what we needed was a gene containing a peptide expressing an interaction with β-lactamase. Using the characteristic that Escherichia c...

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 opened the peptides which can inhibit the ª‰ ¿Clactamases and the gene which can encode the peptides; also it provided the method to prepar the peptides by the expressing carrier. The gene is made up of the 72 nucleotides which encode the 24 amino acids whose mass is 2600Da. The peptides expressed in the E.Coli by the gene can inhibit the ª‰ ¿Clactamases. So it can cure the bacteria infection with the ª‰-lactam Antibiotics.

Description

technical field [0001] The present invention relates to a polypeptide, a gene encoding the polypeptide, and a method for preparing the polypeptide using an expression vector, specifically, a gene encoding a β-lactamase inhibitory polypeptide, and a method for preparing the polypeptide using an expression vector Methods, and pharmaceutical uses of said polypeptides. Background technique [0002] β-lactam antibiotics (penicillins and cephalosporins) are clinically used to treat a variety of bacterial infections, but with the widespread use of antibiotics and abuse to some extent, drug-resistant bacteria are constantly emerging, and they are Resistance to many β-lactam antibiotics renders such drugs ineffective against disease caused by resistant bacteria. Clavulanic acid, an inhibitor of microbial-derived β-lactamase, and its β-lactam sulfone compounds, Sulbactam and Tazobactam, were developed in the 1970s and 1980s. It was discovered and proved to be an effective inhibitor ...

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): A61K38/16A61P31/04C12N9/86C12N15/55C12N15/63C12N15/70C12P21/02
Inventor 朱宝泉孙伟龚家玮朱春宝胡又佳
Owner SHANGHAI INST OF PHARMA IND
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