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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
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  • 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 completely destroying the expression of the gene. If the inserted gene is a relatively important functional gene, this change will have a major impact on cell function. damage, and even cause cell transformation to produce cancer
Using DNA-based plasmids or viruses as tools cannot avoid the related cancer risk in theory, which is also an important reason why gene therapy methods have not been fully promoted

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0114] (1) According to the HDAC6 gene sequence, 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 a template, 6-272 amino acids of HDAC6-Cat1 were amplified. 25 μL of PCR reaction system, containing 12.5 μL of 2× PCR Mix (containing enzyme), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of DNA template, and 9.5 μL of double distilled water. The cycle parameters of PCR were: 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; final extension at 72°C for 10min. 1% agarose gel electrophoresis observation. (3) Construct the pET32a-Cat1-JD recombinant plasmid,...

Embodiment 2

[0115] Example 2: Construction of the HDAC6-CAT1 nanobody library:

[0116] (1) Mix 1 mg of CAT1-JD recombinant protein antigen with 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 nanobodies (2) After 7 times of immunization, extract 100ml of camel peripheral blood lymphocytes, and carry out ELISA antibody level detection of immunized camels, 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 amplify VHH by nested PCR; (4) digest 20ug pMECS phage with restriction enzymes PstⅠ and NotⅠ Display the vector and 10ug VHH and connect the two fragments; (5) Transform the ligation product into electroporation competent cells TG1, construct the human antib...

Embodiment 3

[0117] Embodiment 3: Nanobody screening process against CAT1-JD recombinant protein:

[0118] (1) Take 200uL of recombinant TG1 cells and culture them in 2×TY medium, add 40uL of helper phage VCSM13 to infect TG1 cells during the period, and culture overnight to amplify the phages, use PEG / NaCl to precipitate the phages the next day, and centrifuge to collect the amplified phages ; (2) Couple 200ug of CAT1-JD recombinant protein dissolved in 100mM pH 8.2 NaHCO3 to a microtiter plate, place it overnight at 4°C, and set up a negative control at the same time; (3) Add 100ul of 3% BSA 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), and reacted at room temperature for 1h; (5) Washed 5 times with PBS+0.05%Tween-20 to wash off the bound phage; (6) Use trypsin with a final concentration of 25mg / ml The phage specifically bound to the Fc fragment of the human antibody will be diss...

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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

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Application Information

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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
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