Escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A technology of Escherichia coli and combined vaccine, applied in the field of synthetic biology, can solve the problem of less vaccine development
Active Publication Date: 2018-11-09
NANKAI UNIV
View PDF4 Cites 1 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
Currently, there are few vaccines developed agai
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
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0112] Gene acquisition
[0113] In the present embodiment, the acquisition derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) from Escherichia coli codon-optimized exotoxin A gene (exotoxin A, GI: 877850); and from Campylobacter jejuni ( Campylobacter jejuni ) and the codon-optimized N-glycosyltransferase gene (undecaprenyl-diphosphooligosaccharide--protein glycotransferase, GI: 905417) of Escherichia coli.
Embodiment 2
[0115] Design of gene deletion primers
[0116] In this example, the λRed recombination system is used to knock out two genes of JM109. In this method, resistance is eliminated every time a gene is knocked out. Below to waaL Taking the gene as an example, the steps of gene knockout are explained in detail. wecA Gene deletion primer design is the same.
[0117] Find the nucleotide sequence of JM109waaL, design primers for deletion and identification of waaL. The deletion primer of waaL is waaL-FRT-chl-FRT-F / R, and the identification primer is S-waaL-F / R. Nucleotide sequences are shown in Table 1-8.
Embodiment 3
[0119] JM109Δ waaL build
[0120] 3.1 Transformation of plasmid pSim
[0121] Wild-type JM109 frozen at -80°C was picked and streaked on a non-resistant LB plate, and cultured overnight at 37°C. The next day, a single clone was picked, inoculated into 5 mL LB medium, and cultured overnight at 37° C., 220 rpm. The next day, transfer to 200ml LB medium according to the inoculum volume of 1%. 37°C, 220rpm, cultivate to OD 600 About 0.6-0.8, ice bath for 20min, 5500rpm, 5min, collect the bacteria in a sterilized 50ml centrifuge tube, 4℃, 5500rpm, centrifuge for 5min, discard the supernatant, and use 50ml ice-bathed sterile 10% Resuspend the bacteria in glycerol, 4°C, 5500rpm, and centrifuge for 5 minutes. Repeat the above operation 3 times. For the last time, use the residual liquid when the supernatant was discarded to resuspend the bacteria, and transfer 80 μL to a new sterile EP tube. Freeze at -80°C.
[0122] Thaw the competent cells frozen at -80°C in ice for 10 minutes...
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
Login to view more
Abstract
The invention discloses an escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application, and relates to a method forconstructing a cell factory for synthesizing O1 serum type glycoprotein conjugate vaccines for neonatal meningitis escherichia coli. O1 antigens are constructed based on a DNA Assembler method by utilizing efficient homologous recombination efficiency of saccharomyces cerevisiae so as to synthesize gene cluster plasmids; the O1 antigens are converted in escherichia coli JM109 to synthesize gene cluster shuttle plasmids, and the plasmids are identified by virtue of lipopolysaccharide extraction, gel electrophoresis and silver staining; waaL and wecA in the JM109 are deleted with the aid of FLP-FRT so as to eliminate the interference of original incomplete O antigens; and pET28a(+) plasmids are reconstructed and induced to synthesize the glycoprotein conjugate vaccines, the glycoprotein ispurified by virtue of an AKTA Primeplus protein purification workstation, and the glycoprotein is identified by virtue of western-blotting. The constructed recombinant escherichia coli provides a novel idea for synthesizing the glycoprotein conjugate vaccines by a biological method.
Description
technical field [0001] The invention belongs to the technical field of synthetic biology and relates to a method for synthesizing a neonatal meningitis-causing E. coli glycoprotein-conjugated vaccine by using recombinant Escherichia coli. More specifically, it relates to an Escherichia coli engineering bacterium for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application. Background technique [0002] Neonatal meningitigenic Escherichia coli (NMEC) belongs to a large group of extraintestinal pathogenic Escherichia coli, mainly infecting newborn infants and people with low immunity, and is an opportunistic pathogen. After colonization through the gastrointestinal tract or respiratory tract mucosa, NMEC can invade the blood circulation system and multiply in large numbers, forming high-concentration bacteremia. After the concentration of bacteremia reaches a certain threshold, NMEC will enter the link of brain infection. B...
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
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more