MRNA-coding nanobody and application thereof

A nanobody, coding technology, applied in applications, recombinant DNA technology, antibody mimics/scaffolds, etc., can solve problems such as destruction, cell transformation to produce cancer, and cell function destruction.

Inactive Publication Date: 2017-07-07
SOUTHEAST UNIV
View PDF10 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Using DNA or virus as a representative of plasmids as a medium to express functional proteins in cells is a common means of gene therapy. Although this method has high transfection and expression efficiency, it cannot avoid the recombination of DNA and the genome of target cells. risk of other diseases
For example, foreign DNA may be inserted into a normal gene, causing mutation or even comple

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
  • MRNA-coding nanobody and application thereof
  • MRNA-coding nanobody and application thereof
  • MRNA-coding nanobody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0113] Example 1: Expression and purification of truncated HDAC6-CAT1 protein:

[0114] (1) According to the gene sequence of HDAC6, use Premier Primer5.0 software to design PCR primers: CAT1-JD-5-sal1 (cgaGTCGACgagcagttaaatgaattccattg) and CAT1-JD-3-not1 (gcgGCGGCCGCggcggccatctcacccttggggtcc), the length of the amplified gene fragment is 801bp; ( 2) Using CAT1-JD-5-sal1 and CAT1-JD-3-not1 as primers and HDAC6-WT recombinant plasmid as template, the 6-272 amino acids of HDAC6-Cat1 were amplified. PCR reaction system 25μL, containing 2× PCR Mix (including enzyme) 12.5μL, upstream primer 1μL, downstream primer 1μL, DNA template 1μL, double distilled water 9.5μL. The cycle parameters of PCR are: pre-denaturation at 95°C for 8min; denaturation at 95°C for 40s, annealing at 57°C for 40s, extension at 72°C for 50s, 35 cycles; and finally at 72°C for 10min. Observe by 1% agarose gel electrophoresis. (3) Construct the pET32a-Cat1-JD recombinant plasmid, the method is as follows: use a ...

Example Embodiment

[0115] Example 2: Construction of Nanobody library against HDAC6-CAT1:

[0116] (1) Mix 1 mg of CAT1-JD recombinant protein antigen and Freund’s adjuvant in equal volumes, and immunize a Xinjiang Bactrian camel once a week for a total of 7 consecutive immunizations. During the immunization process, B cells are stimulated to express specific nanoantibodies. (2) After 7 immunizations, 100ml of peripheral blood lymphocytes of camels were extracted, and the ELISA antibody levels of immunized camels were tested. The results are as follows image 3 As shown, the serum titer after immunization reached 10 4 , Indicating that the HDAC6-CAT1-JD recombinant protein has a better immune effect; and extract total RNA; (3) synthesize cDNA and use nested PCR to amplify VHH; (4) use restriction enzymes Pst I and Not I to digest 20ug pMECS phage Display vector and 10ug VHH and connect the two fragments; (5) Transform the ligation product into electro-competent cells TG1, construct a human antibody ...

Example Embodiment

[0117] Example 3: Nanobody screening process for CAT1-JD recombinant protein:

[0118] (1) Take 200uL of recombinant TG1 cells and culture them in 2×TY medium, add 40uL helper phage VCSM13 to infect TG1 cells, and culture overnight to amplify the phages. The next day, use PEG / NaCl to precipitate the phages and collect the amplified phages by centrifugation. (2) 200ug of CAT1-JD recombinant protein dissolved in 100mM pH 8.2 NaHCO3 was coupled to an enzyme-labeled plate, placed overnight at 4℃, and a negative control was set up; (3) 100ul of 3% BSA was added the next day, Block at room temperature for 2h; (4) After 2h, add 100ul amplified phage (2×10 11 tfu immunized camel nanobody phage display gene library), reacted at room temperature for 1h; (5) Washed 5 times with PBS+0.05% Tween-20 to wash off the bound phage; (6) Trypsin with a final concentration of 25mg / ml The phage that specifically binds to the human antibody Fc fragment will be dissociated and infected with E. coli TG1 ...

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 an mRNA-coding nanobody and application thereof, belongs to the field of biomedicine, and particularly relates to the field of nanobody drugs. In order to solve the problems, a new thought and method system is provided for regulating intracellular proteins by using the RNA-coding nanobody. According to the mRNA-coding nanobody provided by the invention, the coded message carried by mRNA is recognized and translated in cells to express a single-stranded nanobody capable of binding to a target protein. The function of disease-related proteins in cells can be effectively and specifically intervened by adopting the method so as to achieve the purpose of treating diseases. Different from the common strategy for expressing nanobodies in cells by using a DNA or virus as a carrier, the method eliminates the risk of altering of the genome sequence of host cells, has the characteristics of safety and easiness in removal, and is very suitable for clinical drug design and production needs.

Description

technical field [0001] The invention relates to a nanobody encoded by mRNA and an application thereof, belonging to the field of biomedicine, in particular to the field of nanobody medicine. Background technique [0002] With the advancement of biomedical research, a large number of protein dysfunctions related to the occurrence of diseases have been discovered, and the vast majority (more than 90%) of the pathogenic proteins are located inside the cell. However, since it is difficult for ordinary monoclonal antibodies to effectively enter the interior of cells, the existing antibody drugs all achieve their therapeutic effects by binding to target proteins located on the cell surface or outside the cells. The number of extracellular target proteins is very limited. Due to this limitation, the target proteins recognized by antibody drugs in the market are highly concentrated. On the one hand, this greatly limits the scope of indications of antibody drugs, and many diseases ...

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): C12N15/13C12N15/85
CPCC07K16/40C07K2317/22C07K2317/569C12N15/85C12N2800/107C07K16/18C07K2319/40C12N15/87C12N15/88
Inventor 谢维郝睿周雪晨周云燕苏志鹏
Owner SOUTHEAST 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