Search

CN-121971660-A - Construction method and application of self-assembled air bag targeting inflammatory factors

CN121971660ACN 121971660 ACN121971660 ACN 121971660ACN-121971660-A

Abstract

The invention discloses a construction method and application of a self-assembled air bag targeting inflammatory factors, the construction method comprises the steps of engineering an air bag gene sequence, enabling air bag outer surface proteins to be fused directionally, expressing short peptide affinity tags, designing a bispecific nanometer antibody gene sequence, co-transforming the air bag genes and the bispecific nanometer antibody genes into host cells to realize self-assembly, crushing the host cells successfully expressing the self-assembled air bag, and obtaining the self-assembled air bag targeting inflammatory factors through centrifugal separation. The self-assembled air bag of biological origin is prepared in a pure biosynthesis mode, the high targeting of inflammatory factors can be realized without carrying out chemical modification on the air bag again, the high targeting of inflammatory factors is realized, the high targeting has good biocompatibility, the specific targeting of inflammatory lesions can be realized, the medical image contrast of the inflammatory sites is obviously enhanced through ultrasonic echo signals generated by internal gas, and the key problem of difficult imaging of the early inflammatory lesions is effectively solved.

Inventors

  • YANG FANG
  • XIONG HAO

Assignees

  • 东南大学

Dates

Publication Date
20260505
Application Date
20260203

Claims (10)

  1. 1. The self-assembled air sac construction method for targeting inflammatory factors is characterized by comprising the following steps: (1) Engineering the airbag gene sequence to ensure that the airbag outer surface protein coded by the modified airbag gene is directionally fused to express the short peptide affinity tag; (2) The gene sequence of the bispecific nano antibody is designed, so that the coded bispecific nano antibody can specifically recognize the short peptide affinity tag and can specifically recognize disease antigens or markers or various inflammatory factors; (3) Transferring the modified airbag gene and the designed bispecific nano antibody gene in the step (1) into a host cell, and realizing the co-expression and self-assembly of the airbag protein expressed by the modified airbag gene and the bispecific nano antibody in the host cell to obtain the host cell successfully expressing the self-assembled airbag; (4) And crushing the host cells successfully expressing the self-assembled air bags, and obtaining the self-assembled air bags targeting inflammatory factors through centrifugal separation.
  2. 2. The method for constructing a self-assembled airbag targeting inflammatory factors according to claim 1, wherein the airbag gene sequence in the step (1) comprises GvpA, gvpA, gvpC, gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and GvpU, wherein the GvpC gene sequence is used as a molecular modification object, a gene sequence of a short peptide affinity tag is inserted into the tail end of GvpC, and the gene sequence and the gene of the short peptide affinity tag are connected through a short hinge, wherein the amino acid sequence corresponding to the GvpC original gene sequence is SEQ ID No. 4, and the amino acid sequence corresponding to the GvpC gene sequence after molecular modification is any one of SEQ ID No. 5-7.
  3. 3. The method for constructing the self-assembled air bag targeting inflammatory factors according to claim 1, wherein the short peptide affinity tag can be recognized by the bispecific nanobody in the step (2), the amino acid sequence corresponding to the short peptide affinity tag is preferably any one of SEQ ID No. 1-3 or other short peptide sequences which can be recognized by a specific nanobody, and the design of the gene sequence of the bispecific nanobody is specifically that the gene sequence of the targeting inflammatory factor nanobody is connected in series with the gene sequence of the targeting short peptide affinity tag nanobody, and the genes of the two nanobodies are connected through a hinge gene.
  4. 4. The method for constructing a self-assembled air bag targeting inflammatory factors according to claim 3, wherein the nano antibody targeting inflammatory factors is a nano antibody targeting interleukin-6 (IL-6), a nano antibody targeting interleukin-17 (IL-17), a nano antibody targeting tumor necrosis factor-alpha (TNF-alpha) or a nano antibody targeting other inflammatory factors, and the amino acid sequence corresponding to the gene sequence of the nano antibody targeting IL-6 is shown in SEQ ID No. 8.
  5. 5. The method for constructing a self-assembled air bag targeting inflammatory factors according to claim 3, wherein the targeting short peptide affinity tag nanobody can be a nanobody targeting SEQ ID No. 1, or SEQ ID No. 2, or SEQ ID No. 3 or other short peptide affinity tags, wherein the amino acid sequence corresponding to the gene sequence of the nanobody targeting SEQ ID No. 1, or SEQ ID No. 2, or SEQ ID No. 3 short peptide affinity tag is shown as SEQ ID No. 9-11, and the hinge gene is IGA LINKER gene, the sequence of which is TCTACTCCGCCGACTCCATCTCCGTCTACTCCGCCG.
  6. 6. A construction method of a self-assembled air bag targeting inflammatory factors is characterized in that the method comprises the following steps of (a) introducing an engineered nano-air bag gene sequence into an expression vector to prepare a recombinant plasmid a, introducing a bispecific nano-antibody gene sequence into the expression vector to prepare a recombinant plasmid b, (b) transforming the recombinant plasmid a obtained in the step (a) into a competent strain, expanding expression to obtain a strain successfully expressing the nano-air bag gene, and preparing the strain again into a competent strain, (c) transforming the recombinant plasmid b obtained in the step (a) into the competent strain in the step (b), expanding expression to obtain a strain successfully expressing the nano-air bag gene and the bispecific nano-antibody gene, d) inducing the strain expressed in the step (c) into the two genes, e) inducing the strain expressed in the step (c) into a suspension strain, and d) suspending the strain after the steps of expression and centrifugation.
  7. 7. The method for constructing the self-assembled air bag targeting the inflammatory factors according to claim 6, wherein the specific method for obtaining the self-assembled air bag targeting the inflammatory factors through centrifugal separation in the step (4) comprises the following steps of (a) centrifuging the cracked host cell lysate at a low temperature, (b) standing the lysate in the step (a), (c) sucking the air bag solution suspended on the upper layer of the lysate in the step (b), and (d) centrifuging the air bag solution in the step (c) at a low temperature and discarding the supernatant component after centrifugation to obtain the self-assembled air bag targeting the inflammatory factors.
  8. 8. A self-assembled inflammatory factor-targeting balloon prepared by the method of constructing a self-assembled inflammatory factor-targeting balloon of claim 1.
  9. 9. Use of the self-assembled inflammatory factor-targeting balloon of claim 8 for the preparation of an ultrasound imaging diagnostic contrast agent for inflammatory lesions.
  10. 10. The use of the self-assembled balloon targeting inflammatory factors in the manufacture of a medicament for ultrasound-mediated inflammatory treatment of claim 8, wherein the self-assembled balloon is targeted to enrich inflammatory factors in a focal site and inactivate the enriched inflammatory factors by using the generated acoustomachanical effect under the action of a physical field of external ultrasound, thereby achieving the regulation and treatment of inflammatory levels.

Description

Construction method and application of self-assembled air bag targeting inflammatory factors Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a construction method and application of a self-assembled air bag targeting inflammatory factors. Background Inflammation has been recognized as a key factor in the onset of various diseases, including ischemic stroke, dilated cardiomyopathy, cancer, and the like. Numerous studies have shown that a variety of diseases are closely associated with uncontrolled release of inflammatory factors and elevated levels of body fluids, including chronic autoimmune diseases (e.g., rheumatoid arthritis, psoriasis, systemic lupus erythematosus, etc.) and severe infectious diseases characterized by cytokine storms (e.g., sepsis, novel coronavirus infection, etc.). The primary feature of sepsis is systemic inflammatory response triggered by immune excess, and recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by immune cells induces secretion of a variety of inflammatory factors, including IFN, IL-6, IL-17A, IL-8, TNF- α, and the like. Monitoring inflammatory factors such as IL-6, IL-17A and TNF- α can identify sepsis when clinical symptoms are not yet apparent, and changes in the levels of these markers often precede the appearance of clinical symptoms. Thus, monitoring of inflammatory factor markers is critical for early diagnosis of sepsis. Current conventional diagnostic methods for inflammation mainly include blood biomarker detection, tissue biopsy, urine analysis, and the like. However, most of these methods can only be tested at a single point in time, and it is difficult to achieve real-time monitoring of the inflammation occurrence and development process. Ultrasound molecular imaging provides a new strategy for this, which enables specific tracking and visualization of target molecules or inflammatory markers by targeted modification of contrast agents. Because of the non-invasive and real-time advantages of ultrasound imaging, it is theoretically possible to dynamically monitor inflammatory processes in real-time. For decades, micron-sized encapsulated bubbles have been used as ultrasound contrast agents, with conventional ultrasound contrast agents mostly in the form of lipid or polymer coated microbubbles. However, such membrane materials have potential biosafety problems due to the use of external substances such as lipids, polymers, and the like. In addition, the size of microbubbles is generally in the micrometer size, larger in particle size, and less stable in vivo, which severely limits their use in molecular imaging of extravascular inflammatory markers. The air sac (GAS VESICLES, GVs) is a protein nanostructure derived from microorganisms such as cyanobacteria, halophilic archaea, and the like. The protein shell layer has the characteristics of hydrophobic inner surface and hydrophilic outer surface, and only allows gas to enter and completely blocks liquid outside, so that the unique hollow inflatable protein nano-airbag is formed. In 2018, the U.S. institute of technology Mikhail G.shape team reported an acoustic reporter gene (Acoustic reporter genes, ARG) for the first time, which was reconstructed from the airbag gene clusters extracted from Bacillus megaterium and Anabaena aquatica, and can encode nanocapsules with uniform particle size and excellent acoustic response performance on the nanoscale, and realize ultrasound imaging of cells and molecular layers. The research team introduces the acoustic reporter gene into the prokaryotic cell E.coli BL21 engineering bacteria and E.coli Nissle 1917 probiotics, both generate GV air bags, and successfully realize ultrasonic imaging of the colon of the mouse with resolution of less than 100 μm by injecting the E.coli Nissle 1917 probiotics expressing GV. The discovery of acoustic reporter genes provides a new direction for the development of ultrasound contrast agents, but the biosynthetic nanocapsules need to be targeted for modification to be applied to ultrasound molecular imaging. The nano antibody is taken as an ideal targeting molecule, is derived from a heavy chain variable region (VHH) of a camelid single-chain antibody, is an antibody functional fragment which is known to be minimum at present and has complete binding activity, and has a molecular weight of only 13-14 kDa, which is about 1/10 of that of a conventional IgG antibody. Besides small volume, the nano antibody has the advantages of high stability, low immunogenicity, easy genetic engineering improvement and the like. At present, although the air sac expressed by the acoustic genes can be used as an ultrasonic contrast agent, the air sac lacks of functional characteristics, and focus targeting cannot be realized. The existing mainstream method needs to carry out additional chemical modification after the extraction of the ai