Chimeric gas vesicle and protein expression system therefor

Abstract

This invention specifically describes a chimeric gas vesicle and its Escherichia coli expression system; the chimeric gas vesicle includes two or more gas vesicle rib proteins, and heteropeptides of 6 to 56 amino acid lengths can be co-inserted into the recombinant rib protein, and the resulting chimeric gas vesicle can be widely used in various applications.

Description

[Technical Field]

[0001] This invention relates to a chimeric gas vesicle and its application. [Previous Technology]

[0002] Gas vesicles are hollow organelles covered by proteins. They are found in the cells of cyanobacteria, soil bacteria, and halobacteria. They are permeable to the surrounding air and provide buoyancy for bacteria to adjust their vertical movement in the aquatic environment in order to obtain sunlight or oxygen.

[0003] The gas vesicles are cylindrical nanostructures with conical ends, ranging from 40 to 250 nanometers in width and 50 to 1000 nanometers in length. These discrete protein nanoparticles, permeable to surrounding air, have considerable and diverse potential applications in bioengineering. In the prior art, the positive increase in buoyancy relying on gas vesicles, the development of acoustic resonance bioimaging, and the expansion of biomass production have been specifically described. Furthermore, as nanoscale biological particles, they are well-suited for vaccine carrier design.

[0004] Note that World Patent Publication No. WO1990010071A1 discloses a recombinant cell and recombinant vector for expressing gas vesicle proteins in *E. coli* and improving the buoyancy of transformed cells. *Bacillus thuringiensis israelensis* (Bti) is well-suited as a biocontrol vector for certain Diptera larvae. When transformed with the gas vesicle gene, *Bacillus thuringiensis* is less prone to aquatic activity and its larvicidal activity is more prolonged. Other hosts incorporating the gas vesicle gene overcome sedimentation issues in bioreactors, promoting the biodegradation of organic waste or medical and agricultural products.

[0005] Note that in World Patent Publication No. WO2018043716A1, Ryota Mizushima and Tomonobu Watanabe jointly published a method for introducing gas vesicle genes, including gvpA and gvpC, into eukaryotic cells to express gas vesicle proteins; these gvpA and gvpC genes are derived from cyanobacteria and have codons optimized for expression in mammals, and are transported into mammalian cells using transposon vectors carrying the gvpA and gvpC genes; the method disclosed in this prior art further provides a kit for establishing stable cell lines or gene-transfected animals that stably express gas vesicles.

[0006] Note that, according to US Patent Publication No. US20140234904A1, Stephen and Levi disclosed a method for collecting photosynthetic single-celled organisms and producing gas vesicles; these self-assembling gas vesicle proteins form conical filaments, which can block water molecules but still allow air to diffuse through; overexpression of these gas vesicles increases the buoyancy of these photosynthetic single-celled organisms and facilitates their harvesting without centrifugation, filtration or chemical flocculation.

[0007] Shapiro et al. (Science 27 Sep 2019: Vol. 365, Issue 6460, pp. 1469-1475) described a specific method for isolating and functionalizing gas vesicles; the nanostructures of the gas vesicles are modified with targeted functional groups and fluorescence, and imaging is performed using ultrasound and magnetic resonance; this method requires 3 to 8 days to prepare these gene-encoding nanostructures, and these nanostructures enable highly sensitive multimodal and non-invasive biological imaging, and have the potential for molecular targeting.

[0008] Note that PCT patent publication number WO2021041934A1, Jin et al., disclose a genetically engineered protease-sensitive gas vesicle; the gas vesicle includes a GvpA or GvpB protein and a genetically engineered GvpC protein; the protease recognition site is inserted into the central block of the GvpC or attached to the N-terminus or C-terminus of the GvpC; these genetically engineered gas vesicles exhibit a nonlinear ultrasonic response from baseline to disintegration; the protease binds to the recognition site and causes the genetically engineered GvpC to break. Subsequently, these genetically engineered gas vesicles disintegrate as the GvpC breaks, and detecting these disintegrations helps to screen genetically engineered protease-sensitive gas vesicles; this method effectively reduces the collapse pressure profile and protease degradation, and improves the performance of genetically engineered gas vesicles in host cells, promoting sufficient protein production; in addition, the ultrasound used for detection and sensing also exhibits enhanced nonlinear behavior.

[0009] Note that US Patent Publication No. US5824309, by DarSarma et al., discloses a method for producing a protein vaccine using recombinant gas vesicles. Foreign antigenic determinants derived from pathogens are inserted into gvpA or gvpC to present antigens, inducing immunity upon administration to a subject. Without adjuvants, the vaccine with recombinant gas vesicles produces a long-lived immunoglobulin response, such as IgG or IgM. In this patent, the archaea *Halobacterium halobium* culture is used as the host cell for the preparation of the genetically engineered gas vesicles.

[0010] Mainstream research on gas vesicles focuses on halobacilli, cyanobacteria, and Bacillus megaterium (Priestia megaterium or Bacillus megaterium).

[0011] In cyanobacteria and halobacteria, the lipid-free, robust protein membrane is composed of the following proteins: GvpA protein, which is mainly a hydrophobic protein with 70 to 85 amino acids, forms the rib-shaped basic structure of gas vesicles, and GvpC protein, which is a minor component and is a hydrophilic protein with 200 to 450 amino acids, as shown in Figure 1. These GvpC proteins are located on the outer surface of the gas vesicles; GvpC is considered a scaffold structural protein because it enhances the structure of the gas vesicles; in the soil bacterium P. megaterium, the GvpB protein with 88 amino acids, which is a homolog of GvpA in cyanobacteria and halobacteria, has been found to be quite necessary for the formation of gas vesicles and plays the same role as GvpA in cyanobacteria and halobacteria. As shown in Figure 6, the 14-gene gvp operon in Bacillus megaterium contains an additional set of gvpA genes, which are homologs of gvpB in Bacillus megaterium, but their function has not yet been determined. Furthermore, no homologs of GvpC have been found in Bacillus megaterium.

[0012] In addition to the gas vesicle main structural protein GvpA and the gas vesicle secondary structural protein GvpC, as shown in Figure 1, five other gas vesicle accessory proteins, GvpJ, GvpM, GvpF, GvpG, and GvpL, were detected by immunoblotting of gas vesicles in *Haemaphysalis*. These accessory proteins constitute only a very small portion of the overall gas vesicle protein composition and are presumed to be located at the conical ends of the gas vesicles, influencing their curvature. Homologous genes for GvpJ, GvpK, and GvpL have been identified in the cyanobacterium *A. flos-aquae*, while a total of five different accessory gas vesicle proteins have been identified in *Bacillus megaterium*.

[0013] Gas vesicle protein A (GvpA) is the main structural protein of gas vesicles, forming the main body of the gas vesicle shell, accounting for about 97% of its mass. The gas vesicle shell is 2 nanometers thick and consists of a single layer of GvpA; GvpA forms 4.6 nanometer "ribs" that are nearly perpendicular to the long axis of the gas vesicle; as shown in Figure 4a, GvpA has a hydrophobic α-helix, β-lamina, and α-helix core amino acid sequence, hereinafter referred to as the GvpA core sequence.

[0014] GvpA proteins are encoded by the gvpA gene and have been sequenced and identified in dozens of microorganisms. These GvpA protein sequences are highly homologous, especially the α-helix, β-fold, β-fold and α-helix core sequences shown in Figure 4a. Only the N-terminal and C-terminal sequences show different patterns in different species.

[0015] Multiple identical copies of the gvpA gene have been found in some cyanobacteria; in halobacteria, two slightly different copies of the gvpA gene have been found in two gvp manipulator sets, located on chromosomes and plastids respectively (c-vac and p-vac), and in the 14-gene gvp manipulator set of Bacillus megaterium (gvpA and gvpB).

[0016] It has long been accepted in the field of gas vesicle research that only one type of rib protein GvpA exists in each gas vesicle; in Halobacterium salinarum, c-vac and p-vac are located in different gene clusters on chromosomes or plastids; the gene expression of C-vac and p-vac is dominated by different mechanisms, and therefore each time a gas vesicle is produced, its rib protein GvpA is expressed from c-vac or p-vac, but no simultaneous expression has been found; in Bacillus megaterium, gvpA and gvpB are located on the same 14-gene gvp operon, but only gvpB is required for the formation of gas vesicles, and the role of gvpA has not yet been clarified.

[0017] Many attempts have been made to modify GvpA by inserting exogenous peptides into the N-terminus, C-terminus, or core sequence of GvpA; however, the rigidity of the GvpA protein limits the space for modification; the DarSarma team revealed that attaching more than 5 heterologous amino acids to the C-terminus of GvpA in H. salinarum will prevent it from forming gas vesicles; other attempts to insert heterologous amino acids into the N-terminus or core sequence have also failed, which limits the applicability of gas vesicles in various projects, such as vaccines, biomarkers and other technologies.

[0018] GvpC is a minor supporting structural protein and has been found on the surface of gas vesicles in cyanobacteria and halophilic bacteria. Taking *Anabaena flos-aquae* (A. flos-aquae) as an example, GvpC accounts for approximately 2.9% of the gas vesicle proteins, and it can detach from the gas vesicle surface under the action of surfactants or changes in salt concentration. As a scaffold protein, GvpC stabilizes the gas vesicle structure through protein-protein interactions with GvpA. In *H. salinarum*, GvpC can attach up to 398 heterologous amino acids, making GvpC more suitable for application in bioengineering technology. These gas vesicles with genetically engineered GvpC are also known as gas vesicle nanoparticles (GVNPs).

[0019] However, the adhesion of GvpC to the surface of gas vesicles is not stable, and the ratio between GvpC / GvpA or GvpC / GV is difficult to maintain at a stable level in practice. For example, GVNPs produced by Haemobacterium can only remain stable in high-salt environments. In low-salt environments, the structure of GVNPs is quite easy to disintegrate, resulting in GvpC detaching from the nanoparticles. Therefore, the structural instability of GVNPs and the tendency of GvpC to detach from the surface of GVNPs reduce the reliability of GVNPs as antigen-presenting carriers or ultrasound and nuclear magnetic resonance imaging biomarkers.

[0020] On the other hand, the production cycle of gas vesicles in halobacteria is as long as 3 to 4 weeks, which is quite time-consuming. Therefore, the production cost of GV or GVNP in halobacteria is always much higher than that of Escherichia coli expression systems. At the same time, there is a lack of suitable vector / host systems in halobacteria to effectively express gas vesicles and produce them in large quantities.

[0021] Therefore, in the attempt to overcome the aforementioned limitations, novel genetically engineered chimeric gas vesicles (CGVs) are urgently needed. [Summary of the Invention]

[0022] In the field of gas vesicle research, it is widely believed that only one protein, GvpA, forms the rib structure of gas vesicles. In other words, a gas vesicle contains only one type of rib protein, such as GvpA from cyanobacteria, halobacteria, and soil bacteria, or the GvpA homolog GvpB from Bacillus megaterium. In this invention, a chimeric gas vesicle is disclosed, which specifically involves two or more rib proteins.

[0023] The chimeric gas vesicles provided by this invention offer several advantages, differing from conventional gas vesicles or gas vesicle nanoparticles. For example, prior art attempts to directly genetically engineer GvpA within gas vesicles ultimately failed due to its inherent rigidity; only a limited number of heteropeptides could insert GvpA without damaging the gas vesicle. The chimeric gas vesicles provided by this invention overcome this limitation, enabling gas vesicles to be more widely applied in the field of biotechnology. In Haematobacterium, genetically modified GvpC in gas vesicle nanoparticles adhere to the gas vesicle surface through protein interactions, making the gas vesicle nanoparticles quite unstable and highly sensitive to temperature and salinity conditions, easily detaching from the gas vesicle surface. In the chimeric gas vesicles provided by this invention, the heteropeptides inserted into the chimeric gas vesicles are covalently bound to ribin, effectively eliminating the issue of heteropeptide detachment from the gas vesicle surface, allowing for smoother presentation by the gas vesicle. Furthermore, the chimeric gas vesicles provided by this invention can be prepared in a relatively short process cycle, significantly shortening the production cycle compared to gas vesicle nanoparticles produced by Haemobacterium or gas vesicles produced by cyanobacteria. Chimeric gas vesicles are more stable than other types of gas vesicles; in other words, the chimeric gas vesicles provided by this invention have a higher "critical disintegration pressure" than conventional gas vesicles. In some cases, these chimeric gas vesicles are quite stable at room temperature and can be maintained for up to one month, while at 4°C they can remain stable for at least six months without degradation.

Implementation Method

[0035] In the field of gas vesicle research, as shown in Figure 1, it is widely believed that only one type of rib protein, namely GvpA, forms the rib structure of gas vesicles; in other words, in a gas vesicle, there is only one type of rib protein, such as GvpA of cyanobacteria, halobacteria and soil bacteria, or GvpB, a homolog of GvpA in Bacillus megaterium.

[0036] In one embodiment of the present invention, a chimeric gas vesicle is provided, comprising at least two gas vesicle rib proteins, wherein the rib proteins are selected from a group consisting of a primary rib protein and a secondary rib protein, wherein the primary rib protein differs from the secondary rib protein in that it has the ability to independently form a gas vesicle; preferably, as shown in Figures 2 and 3, the primary rib protein and the secondary rib protein have only less than 95% homology, wherein any of the gas vesicle rib proteins is derived from P. megaterium.

[0037] In some examples, the at least two gas vesicle rib proteins may be two principal rib proteins, a secondary rib protein, or a combination of a principal rib protein and a primary rib protein; preferably, the secondary rib protein forms a complete chimeric gas vesicle in the presence of the principal rib protein, in which case the chimeric gas vesicle is more stable.

[0038] In several embodiments, the primary rib protein and the secondary rib protein constitute at least 90% of the dry weight of the chimeric gas vesicle.

[0039] In several embodiments, the secondary rib protein includes a core sequence and a heteropeptide, wherein the heteropeptide is in-frame inserted at the C-terminus or N-terminus of the core sequence; specifically, the core sequence is characterized by a protein structure having a first α-helix, a first β-fold located at the C-terminus of the first α-helix, a second β-fold located at the C-terminus of the first β-fold, and a second α-helix located at the C-terminus of the second β-fold; in short, the protein structure is a tertiary structure of α-helix-β-fold-β-helix-α-helix.

[0040] In a preferred embodiment, the core sequence is selected from the group consisting of the GvpA core sequence and the GvpB core sequence.

[0041] In several embodiments, the first α-helix contains the amino acid sequence SSSLAEVIDRILD or SSGLAEVLDRVLD; the first β-fold contains the amino acid sequence KGIVIDAFARVSL, KGIVIDAFARVSV, KGIVIDAWVRVSL, KGVVVDVWARISL, or KGVVVDVWARVSL; the second β-fold contains the amino acid sequence VGIEILTIEARVVI, VGIELLAIEARIVI, or VGIEILTVEARVVA; the second α-helix contains the amino acid sequence ASVDTWLRYAEXZ, ASVVETYLKYAEXZ, or ASVDTFLHYAEXZ; preferably, the X amino acid residue is alanine (Ala) or glutamic acid (Glu), and the Y amino acid residue is valine (Val) or isoleucine (Ile).

[0042] Specifically, the core sequence is a hydrophobic region of the GvpA or GvpB protein; as shown in Figure 4a, the core sequence is a tertiary protein structure composed of four secondary structures in the order of α-helix-β-lamina-β-lamina-α-helix; the GvpA or GvpB protein is derived from cyanobacteria, halobacteria, or soil bacteria; preferably, the soil bacteria is Priestia Megaterium; the halobacteria is Halobacterium salinarum; and the cyanobacteria is Anabaena flos-aquae.

[0043] In some preferred embodiments, the GvpA protein has an amino acid sequence as shown in SEQ ID NO.: 1; and the GvpB protein has an amino acid sequence as shown in SEQ ID NO.: 2.

[0044] Specifically, the core sequence includes, but is not limited to, amino acids 9 to 61 of the GvpA or GvpB protein from Bacillus megaterium; in addition, all gas vesicle rib proteins carry the core sequence; furthermore, any homolog of the GvpA or GvpB protein from cyanobacteria, halobacteria, or soil bacteria, or any protein sequence homologous to the core sequence, can be genetically engineered and serve as the rib protein.

[0045] It should be noted that the aforementioned secondary structure was predicted by the online protein secondary structure prediction program Jpred 4; as shown in Figure 4a, the region forming the α-helix is ​​marked with the underline H, while the region forming the β-fold is marked with S.

[0046] Figure 4b shows a sequence alignment diagram of a secondary rib protein with an exogenous peptide, which roughly shows the design of the secondary rib protein, wherein the exogenous peptide is inserted at the C-terminus of the core sequence.

[0047] In other embodiments, the exogenous peptide has at least 6 amino acids and is derived from artificially designed peptide sequences, receptors, hormones, cytokines, receptors, antibody complementary sites, toxic proteins or fluorescent proteins.

[0048] In some preferred embodiments, the primary rib protein is a GvpB protein, and the secondary rib protein has a core sequence of an exogenous peptide, wherein the exogenous peptide has at least 6 amino acids and the exogenous peptide is inserted at the C-terminus or N-terminus of the core sequence.

[0049] Preferably, the core sequence includes a truncated ribin that retains the first (methionine) to the 61st (valine) amino acid residues of the GvpA or GvpB protein derived from Bacillus megaterium.

[0050] For example, the exogenous peptide can be inserted into the C-terminus of the core sequence, which has 6 to 56 or more amino acid residues, and the core sequence is a truncated form of the gas vesicle rib protein; the truncated gas vesicle rib protein and the exogenous peptide together form the secondary rib protein, which carries an exogenous peptide and can be used in a variety of biotechnology fields; however, a longer exogenous peptide will cause the chimeric gas vesicle to be unstable and reduce its yield.

[0051] In some embodiments, the exogenous peptide may also be derived from two or more exogenous proteins; for example, 20 amino acids from protein A and 25 amino acids from protein B together form an exogenous peptide of 45 amino acids in length, which serves as an insert for forming the secondary rib protein; preferably, the exogenous peptide is derived from the protein sequence of a pathogen, the variant region of an antibody, a hormone, a cytokine, or a receptor.

[0052] In several embodiments, the chimeric gas vesicles are derived from and purified from Escherichia coli (E. coli).

[0053] In one or more embodiments, the chimeric gas vesicle further includes an assembly protein, wherein the assembly protein includes GvpP protein, GvpQ protein or a combination thereof; the assembly protein is derived from Bacillus megaterium; the assembly proteins promote, accelerate or stabilize the formation of the chimeric gas vesicle; with the presence of GvpP protein and GvpQ protein, the subribin can be more uniformly distributed on the surface of the chimeric gas vesicle; the chimeric gas vesicles formed with the assistance of GvpP protein and GvpQ protein are more stable than chimeric gas vesicles without the assistance of GvpP protein and GvpQ protein.

[0054] In some specific embodiments, the GvpP protein has an amino acid sequence as shown in SEQ ID NO.: 3; the GvpQ protein has an amino acid sequence as shown in SEQ ID NO.: 4.

[0055] In a preferred embodiment, the chimeric gas vesicle further includes at least one accessory protein selected from the group consisting of GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU.

[0056] The term "chimeric gas vesicles (CGVs)" means that these gas vesicles are composed of at least two Gvp proteins with different protein sequences; in contrast, gas vesicles described in other published literature involve only one major rib protein, namely GvpA.

[0057] The term "rib protein" refers to the "ribs" that form the gas vesicles or chimeric gas vesicles formed by the gas vesicle protein. Once the gas vesicle is formed, the rib protein cannot be removed from the gas vesicle without destroying it. The gas vesicle rib protein forms "ribs" of about 4.6 nanometers, which are stacked in an array and are nearly perpendicular to the long axis of the gas vesicle, and constitute the main part (97%) of the gas vesicle wall. As shown in Figure 1, these walls are 2 nanometers thick and are composed of a single layer of rib protein. All gas vesicle rib proteins have a hydrophobic tertiary structure with four secondary structures: α-helix, β-laminar, β-laminar and α-helix. As shown in Figures 4a to 4b, the protein sequence of the tertiary structure is defined as the "core sequence".

[0058] The term "master rib protein" refers to GvpA protein from cyanobacteria, halobacteria, and soil bacteria, as well as GvpB from Bacillus megaterium, or any homologous Gvp protein that functions equivalent to GvpA protein, which can form the rib structure of gas vesicles; the gas vesicle master rib protein is a rib protein with a "core sequence"; together with trace amounts of Gvp accessory structural proteins such as GvpF, GvpG, GvpJ, GvpK, GvpL, and GvpS, the master rib protein forms a complete gas vesicle; these master rib proteins are sufficient to form gas vesicles without involving other rib proteins; once the gas vesicle is formed, these master rib proteins cannot be removed from the gas vesicle without destroying it; these gas vesicle master rib proteins constitute 20% to 99.9% of the total gas vesicle protein, and are considered to be master structural proteins, master constituent proteins, rib proteins, etc.

[0059] The term "secondary rib protein" refers to a rib protein with a "core sequence" that forms gas vesicles only in the presence of another rib protein. Most of the time, the other rib protein is the gas vesicle master rib protein, but it can also be another gas vesicle secondary rib protein with a different protein sequence. Once the chimeric gas vesicle is formed, it cannot be removed from the chimeric gas vesicle without destroying it. These gas vesicle secondary rib proteins constitute 3 to 80% of the total gas vesicle protein.

[0060] Natural rib proteins are gas vesicle rib proteins, GvpA or their homologs found in the natural environment; truncated rib proteins are natural gas vesicle rib proteins with some amino acid sequences removed; these recombinant rib proteins are natural or truncated gas vesicle rib proteins with exogenous peptides inserted into the C-terminus, N-terminus or mid-segment.

[0061] The so-called "core sequence" refers to the hydrophobic region of GvpA or its homologs, as shown in Figures 4a to 4b, which has a tertiary structure consisting of four secondary structures: α-helix, β-laminar, β-laminar and α-helix; these "core sequences" span from the 9th (serine) to the 61st (valine) amino acid residues of GvpA or GvpB derived from Bacillus megaterium; all gas vesicle rib proteins carry this core sequence.

[0062] On the other hand, the present invention provides a protein expression system for expressing the chimeric gas vesicle, wherein the protein expression system includes a first polynucleotide fragment encoding one of the gas vesicle rib proteins and a second polynucleotide fragment encoding another gas vesicle rib protein; wherein the protein expression system may be a DNA-based protein expression system or an RNA-based protein expression system.

[0063] In several embodiments, the first polynucleotide fragment encodes the major rib protein; the second polynucleotide fragment encodes the minor rib protein, wherein the second polynucleotide fragment includes a core polynucleotide fragment encoding a core sequence and a heteropolynucleotide fragment encoding a heteropeptide.

[0064] In some preferred embodiments, the core sequence is characterized by a tertiary structure comprising a first α-helix; a first β-bend located at the C-end of the first α-helix; a second β-bend located at the C-end of the first β-bend; and a second α-helix located at the C-end of the second β-bend; preferably, the core sequence is selected from a group consisting of a GvpA core sequence and a GvpB core sequence.

[0065] Specifically, as shown in Figure 4a, the core sequence is the hydrophobic region of the GvpA or GvpB protein, wherein the core sequence contains the 9th to 61st amino acids of the GvpA or GvpB protein; preferably, the core sequence is a protein tertiary structure having four protein secondary structures arranged in the following order: α-helix, β-lamella, β-lamella and α-helix, wherein the GvpA or GvpB protein is derived from cyanobacteria, halobacteria or soil bacteria; preferably, the soil bacteria is P. megaterium; the halobacteria is H. salinarum; the cyanobacteria is A. flos-aquae.

[0066] In a preferred embodiment, the first polynucleotide sequence has a nucleotide sequence such as SEQ ID NO.: 5 or SEQ ID NO.: 6.

[0067] In several embodiments, the exogenous peptide has at least 6 amino acids, wherein the exogenous peptide is derived from a natural peptide sequence selected from the group consisting of ligands, hormones, cytokines, receptors, paratopes of antibodies, toxic proteins, and fluorescent proteins, or an artificially designed peptide sequence.

[0068] In some embodiments, the protein expression system further includes an assembly polynucleotide fragment encoding an assembly protein, wherein the assembly protein includes GvpP protein, GvpQ protein, or a combination thereof; with the assembly protein, the secondary rib protein can be more evenly distributed on the surface of the chimeric gas vesicle; compared with chimeric gas vesicles assisted by the assembly protein, if GvpP protein or GvpQ protein is missing, the chimeric gas vesicles appear to be less stable and prone to assembly errors or reduced yield.

[0069] In some specific embodiments, the assembled polynucleotide fragment is derived from gvpP, gvpQ or a combination thereof of Bacillus megaterium; preferably, the assembled polynucleotide fragment has a nucleotide sequence such as SEQ ID NO.: 7, SEQ ID NO.: 8 or SEQ ID NO.: 9.

[0070] In some embodiments, the protein expression system further includes an accessory polynucleotide fragment encoding at least one accessory protein selected from the group consisting of GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT, and GvpU; in some specific embodiments, the accessory polynucleotide fragment has a nucleotide sequence as shown in SEQ ID NO.: 10.

[0071] In some embodiments, the protein expression system may be induced to express the gas vesicle proteins in a host, wherein the host includes archaea, prokaryotic cells, or eukaryotic cells; preferably, the archaea is H. salinarum, the prokaryotic cell is Escherichia coli, and the eukaryotic cell is mammalian cells, yeast, or insect cells; more preferably, the host is Escherichia coli.

[0072] As shown in Figure 8a, these gas vesicles are purified self-transformed *Escherichia coli* containing the *Bacillus megaterium* gene, which possesses gvpB (encoding a master rib protein) and gvpRNFGLSKJTU; these gas vesicles are mostly small (40 x 40 nm) and some are large (40 x 200 nm). (nm); In some exemplary examples, as shown in Figure 8b, the chimeric gas vesicles of *E. coli* that have been purified from self-transformed giant spore-forming bacteria genes have gvpA (encoding recombinant secondary rib protein), gvpPQ (encoding assembly protein), gvpB (encoding primary rib protein), and gvpRNFGLSKJTU; with these two gas vesicle rib proteins and the assembly protein, these chimeric gas vesicles exhibit a large size (40x200nm); in the above example, the secondary rib protein is a 77-amino acid GvpA protein fused with an exogenous peptide of 17 amino acids. Table 1 plastid name C-end deletion (AA) Heteropeptides (AA) GvpA C-terminal preserved sequence CGV production (μg / ml per culture) pNL39 0 0 WLRYAEAVGLLTDKVEEEGLPGRTEERGAGLSF 10 pNL114 6 0 WLRYAEAVGLLTDKVEEEGLPGRTEER 10 pNL115 10 0 WLRYAEAVGLLTDKVEEEGLPGR 10 pNL119 13 0 WLRYAEAVGLLTDKVEEEGL 10 pNL118 16 0 WLRYAEAVGLLTDKVEE 10 pNL105 18 0 WLRYAEAVGLLTDKV 10 pNL106 19 0 WLRYAEAVGLLTDK 10 pNL107 20 0 WLRYAEAVGLLTD 10 pNL131 22 0 WLRYAEAVGLL 10 pNL132 24 0 WLRYAEAVG 10 pNL135 25 0 WLRYAEAV 10 pNL133 26 0 WLRYAEA N / A pNL136 27 0 WLRYAE N / A pNL134 28 0 WLRYA N / A pNL151 25 47 WLRYAEAV + 47-AA 6 to 8 pNL148 25 48 WLRYAEAV + 48-AA 6 to 8 pNL149 25 49 WLRYAEAV + 49-AA 6 to 8 pNL150 25 50 WLRYAEAV + 50-AA 6 to 8 pNL162 25 53 WLRYAEAV + 53-AA 1 to 2 pNL163 25 56 WLRYAEAV + 56-AA 1 to 2

[0073] In some embodiments, in order to reduce the impact of longer heteropeptides, the number of amino acids deleted by these gas vesicle proteins must also be taken into consideration; without affecting the assembly and formation of chimeric gas vesicles, a maximum of 25 amino acids can be deleted at the C-terminus of GvpA and a maximum of 27 amino acids can be deleted at the C-terminus of GvpB; in one example, the maximum value of the peptide fragment that GvpA can be truncated and the maximum value of the heteropeptide that can be inserted are defined; as shown in Table 1, without destroying the chimeric gas vesicles, a heteropeptide with up to 25 amino acids can be removed from Bacillus megaterium GvpA and has at least 56 amino acids can be inserted into the C-terminus of the truncated GvpA; in addition, it can be understood from Table 1 that when a shorter heteropeptide needs to be inserted, fewer amino acids can be deleted at the C-terminus of the protein GvpA or GvpB.

[0074] In other embodiments, the protein expression system can be implemented using two or more different expression vectors to enable the chimeric gas vesicles to be expressed in a host; in one example, a polynucleotide fragment encoding GvpA, GvpP, and GvpQ is inserted into a first vector, while another polynucleotide fragment encoding GvpB, GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT, and GvpU is inserted into a second vector; the first vector and the second vector are co-transformed into a host to produce the chimeric gas vesicles; preferably, when the host is Escherichia coli, these vectors are derived from plasmid pST39 and plasmid pLysS.

[0075] The term "vector" refers to a nucleic acid molecule that, for a specific biological function such as protein expression or amplification of the copy number of a target gene, includes gene expression elements and gene engineering elements to carry the target gene into a selected host.

[0076] In a preferred embodiment, as shown in FIG5, in order to construct a gvp operon system, which includes a single operon that can represent the chimeric gas vesicle, the 3' end of the gvpAPQ polynucleotide fragment or the gvpBPQ polynucleotide fragment is covalently linked to the 5' end of the gvpBRNFGLSKJTU polynucleotide fragment to form a gvpAPQBRNFGLSKJTU polynucleotide fragment or a gvpBPQBRNFGLSKJTU polynucleotide fragment.

[0077] As shown in Figure 6, in order to establish the gvp operon system, it has two operons that can respectively represent the major rib protein and the minor rib protein. The 3' end of the second polynucleotide fragment is covalently linked to the 5' end of the assembled polynucleotide fragment to form a gvpAPQ polynucleotide fragment or a gvpBPQ polynucleotide fragment, or the 3' end of the first polynucleotide fragment is covalently linked to the 5' end of the auxiliary polynucleotide fragment to form a gvpARNFGLSKJTU polynucleotide fragment or a gvpBRNFGLSKJTU polynucleotide fragment.

[0078] Specifically, an operator or a promoter is positioned upstream of the gvpAPQ polynucleotide fragment, the gvpBPQ polynucleotide fragment, the gvpBRNFGLSKJTU polynucleotide fragment, the gvpAPQBRNFGLSKJTU polynucleotide fragment, and the gvpBPQBRNFGLSKJTU polynucleotide fragment, so that the gvp operon system can synthesize the protein required for the assembly of chimeric gas vesicles under the control of an inducing factor, such as isopropyl thiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside, phenyl-β-D-galactose, or o-nitrophenyl-β-galactoside (ONPG), but is not limited thereto.

[0079] In a preferred embodiment, the gvpAPQBRNFGLSKJTU polynucleotide fragment has a DNA sequence of 7053 base pairs, encoding GvpA, GvpP, GvpQ, GvpB, GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT, and GvpU, and can be inserted into a suitable expression vector to achieve the protein expression of chimeric gas vesicles; preferably, the gvpAPQBRNFGLSKJTU polynucleotide fragment has a nucleotide sequence as shown in SEQ ID NO.: 11.

[0080] Another embodiment of the present invention provides a method for producing chimeric gas vesicles, wherein the chimeric gas vesicles have a genetically modified gas vesicle secondary rib protein GvpA or GvpB fused with a heterologous peptide, as shown in Figure 9. The method includes the following steps:

[0081] (Step S1) Plastosome construction: A DNA sequence encoding a heteropeptide of 6 to 56 amino acids or longer is inserted into the C-terminus and / or N-terminus of a genetically modified GvpA or GvpB protein by polymerase chain reaction (PCR) and Gibson Assembly in-frame to construct a plastome, wherein the GvpA or GvpB is located in the recombinant gvp operon and is derived from Bacillus megaterium;

[0082] (Step S2) Plastid transformation: The aforementioned constructed plasmids are transformed into Escherichia coli or other suitable bacterial hosts, which have the ability to express and produce chimeric gas vesicles;

[0083] (Step S3) Induction of expression: The aforementioned transgenic strain is cultured in a suitable environment to the optimal conditions for inducing protein expression; the aforementioned transgenic strain is stimulated to express the chimeric gas vesicles with IPTG or other inducing factors;

[0084] (Step S4) Purify and quantify the chimeric gas vesicle.

[0085] To further confirm the sequence of the recombinant manipulation group, the method further includes the following steps:

[0086] (Step S1.1) Sequence verification: After step S1, the plasmid sequence with the expected insert is confirmed by DNA sequencing.

[0087] In some embodiments, step S1 includes inserting a DNA sequence encoding the subribin into a gvp operon vector by PCR and Gibson Assembly to construct a chimeric gas vesicle expressplast, wherein the subribin has a heteropeptide of at least 6 amino acids in the same frame.

[0088] In another embodiment of the present invention, a method for eliciting an immune response is provided, comprising administering CGV-Covid-19 to a desired individual, as shown in FIG10, the method comprising the steps of:

[0089] (Step A) Purify and quantify the CGV-Covid-19 vaccine in phosphate-buffered saline (PBS);

[0090] (Step B) Administer the CGV-Covid-19 vaccine to the individual in need; and

[0091] (Step C) Measure the immune response of the desired individual.

[0092] In a particular embodiment, CGV-Covid-19 is administered to the desired individual via intranasal delivery, wherein a heteropeptide of 17 amino acids, derived from the Covid-19 virus S protein, is inserted at the C-terminus of the core sequence, thereby presenting an antigenic determinant derived from the SARS-CoV-2 spike protein in the chimeric gas vesicles; here, an enzyme-linked immunosorbent assay (ELISA) is used to measure the immune response elicited by the CGV-Covid-19 vaccine, but the assay method is not limited thereto; the desired individual may include, for example, humans, mice, dogs, dolphins, chimpanzees or other mammals.

[0093] The chimeric gas vesicles provided by the present invention can be applied to vaccines for infectious diseases, therapeutic vaccines for cancer or other diseases; the chimeric gas vesicles can be used as therapeutic agents for cancer, metabolic diseases or other diseases, such as carrying hormones, cytokines, receptors or partial antibody variants; they can also be used as invasive contrast agents for ultrasound, MRI or other imaging techniques; in short, the chimeric gas vesicles as nanoparticles have the potential advantage of wide application.

[0094] Experimental Example 1: Plastid Construction and Validation Any applicable Escherichia coli plasmid / host expression system can be used, such as pBB322-derived plasmid / BL21 Escherichia coli strain (New England Biolab), pET28 plasmid / BL21 (A1) Escherichia coli strain (Thermo Fisher), or pST39 plasmid / Rosetta Escherichia coli strain (Millipore Sigma).

[0095] The plastid is digested with restriction enzymes or amplified by PCR with a specific primer to produce the corresponding DNA fragment; the DNA primer encoding the target peptide can be designed by someone with ordinary skills in the art or purchased from any commercial primer supplier.

[0096] The aforementioned DNA fragment encodes a heterologous truncated GvpA or GvpB protein and a target peptide, and a DNA fragment carries the remaining 13 gvp genes: gvpPQBRNFGLSKJTU derived from Bacillus megaterium, which is obtained by PCR amplification using a suitable DNA primer; these fragments are then joined together with Gibson Assembly (Thermo Fisher), and these Gibson Assembly mixtures are transduced into suitable Escherichia coli competitive cells, which can then be analyzed and sequenced by anyone with ordinary skill in the art to confirm these transduced strains.

[0097] Characteristics and Production of Chimeric Gas Vesicles

[0098] Plasmids with the correct DNA sequence insert were transduced into the intended *E. coli* host, and single colonies were selected and inoculated into LB medium with appropriate selective antibiotics; chimeric gas vesicles were induced to appear with IPTG or other inducing factors and purified using a modified experimental procedure comprising the following steps: a. After culturing the transduced *E. coli* for 20 to 22 hours, centrifuged at 500g for 1 to 2 hours; b. The precipitate was dispersed with phosphate-buffered saline (PBS) and lysed with surfactant, lysozyme, and deoxyribonuclease; c. The cell lysate was then centrifuged at 500g for 5 to 30 minutes to separate the chimeric gas vesicles from the cell debris; it should be noted that the chimeric gas vesicles floated on top of the lysate suspension; d. Cell debris and midnatant were removed with a needle; e. f. Disperse the preserved chimeric gas vesicles in PBS; g. Repeat steps c to e ten times until the purity of the chimeric gas vesicles reaches 99%; g. Measure the absorbance (OD500) of the chimeric gas vesicles at wavelength 500 nm using a spectrophotometer (NanoDrop ND-1000, Thermo Scientific) to calculate its final concentration; it should be noted that 1 unit of OD500 of the chimeric gas vesicle is equivalent to 145 ug / ml of protein.

[0099] Experimental Example 2 To verify that the present invention can successfully insert heteropeptides derived from one or more foreign proteins, in Experimental Example 2, a peptide of 20 amino acids in length derived from hemagglutinin (HA) of H1N1 virus and a spike protein derived from Covid-19 virus were combined to form a peptide insert of 40 amino acids in length; then, the aforementioned heteropeptide was inserted into the 14-gene gvp operon, which may be on the same or two different plastids; the plastids were transformed or co-transformed into competent E. coli cells to produce chimeric gas vesicles carrying two or more heteropeptides in large quantities.

[0100] Experimental Example 3 In order to further verify the protein expression of chimeric gas vesicles carrying multiple heteropeptides, the chimeric gas vesicles produced according to the method and steps of Experimental Example 1 were confirmed by Western dot blotting.

[0101] Chimeric gas vesicles containing multiple heteropeptides were purified from E. coli cultures, and their transformation resulted in corresponding plastid structures; Western speckle staining was performed using rabbit anti-Covid-19 S protein as the primary antibody and goat anti-rabbit IgG-HRP as the secondary antibody. Table 2 Ink dot numbering CGV sample Heteropeptide sequences S protein sequence 1 Positive control group Covid-19 S protein S protein 2 pNL39 GvpA without inserted heteropeptide sequence Heteropeptide sequence 3 pNL66 TGCVIAWNSNNLDS T430 to S443 4 pNL67 NLDSKVGGNYNYLY N440 to Y453 5 pNL68 NYLYRLFRKSNLKP N450 to P463 6 pNL69 NLKPFERDISTEIY N460 to Y473 7 pNL70 TEIYQAGSTPCNGV T470 to V483 8 pNL71 CNGVEGFNCYFPLQ C480 to Q493 9 pNL72 FPLQSYGFQPTNGV F490 to V503 10 pNL73 TNGVGYQPYRVVVL T500 to S513

[0102] As shown in Figure 7, the positive ink dots representing chimeric gas vesicles are ink dots numbered 5 to 8, which show that the antigen recognition site of the Covid-19 S protein is located at amino acid residues 450 to 493; Table 2 lists the Covid-19 S protein sequence as a recombinant gvp operon insert in more detail, and the antigen sites of the Covid-19 S protein that can be recognized are NYLYRLFRKSNLKP (N450 to P463), NLKPFERDISTEIY (N460 to Y473), TEIYQAGSTPCNGV (T470 to V483) and CNGVEGFNCYFPLQ (C480 to Q493).

[0103] The chimeric gas vesicles (CGVs) provided by the present invention have the following specific advantages over conventional gas vesicles (GVs) and gas vesicle nanoparticles (GVNPs).

[0104] For example, due to the rigidity of GvpA, direct genetic modification of GvpA in conventional gas vesicles has not been successful in the past. To date, only heteropeptides with a length of 5 amino acids have been successfully inserted into the C-terminus of GvpA without disrupting the formation of the gas vesicle. The chimeric gas vesicles disclosed in this invention overcome the limitation of stability and open up possibilities for various applications of chimeric gas vesicles. In past studies of Haematobacterium gas vesicles, the genetically modified GvpC in GVNPs only attaches to the surface of gas vesicles through protein-protein interactions, making it quite sensitive and fragile to temperature and salinity changes. GvpC is easily detached from GVNPs, greatly reducing the applicability of conventional gas vesicles. In chimeric gas vesicles, these heteropeptides can be inserted into natural GvpA, truncated GvpA, or their homologs, or covalently fused. The chimeric gas vesicles fuse with the rib proteins in a fused form to form these rib proteins; as part of the rib structure, the problem of heteropeptide shedding faced by conventional gas vesicles is solved; for industrial or commercial production, the production cycle of chimeric gas vesicles is significantly shortened; in some cases, the production cycle can be shortened to less than 2 days, far shorter than the production cycle of GVNPs in Haematobacterium or gas vesicles in Cyanobacteria; chimeric gas vesicles are more stable than conventional gas vesicles; chimeric gas vesicles produced by Escherichia coli exhibiting GVP operative groups derived from Bacillus megaterium are more stable than any known gas vesicles; compared to conventional gas vesicles, the chimeric gas vesicles provided by this invention have a higher "critical collapse pressure". Regarding storage, in one embodiment, the chimeric gas vesicles maintained their integrity for at least one month at room temperature and for up to six months at 4°C without any degradation. Based on the above characteristics exhibited by the chimeric gas vesicles, the present invention significantly enhances the future applicability of chimeric gas vesicles.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials to those described and used herein may be used in the implementation or testing of this invention, preferred methods and materials are as described above. All publications, patent applications, publications, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to limit the scope of protection declared in this invention. Steps: S1 to S4 Step: S1.1 [Simplified Explanation of the Diagram]

[0024] Figure 1 is a cartoon diagram used to illustrate the known structure of natural gas vesicles.

[0025] Figure 2 is a cartoon diagram used to illustrate the chimeric gas vesicles provided by the present invention.

[0026] Figure 3 is a cartoon diagram used to illustrate chimeric gas vesicles with heteropeptides.

[0027] Figure 4a illustrates the sequence alignment results of the gas vesicle protein.

[0028] Figure 4b illustrates the sequence alignment results of recombinant secondary rib protein with heteropeptide.

[0029] Figures 5 and 6 show the gene manipulation of the chimeric gas vesicle gvp operative group.

[0030] Figure 7 is a Western ink dot pattern used to confirm that chimeric gas vesicles carry different types of heteropeptides.

[0031] Figure 8a is an electron microscope image of a gas vesicle negatively stained, with a scale bar of 200 nanometers.

[0032] Figure 8b is an electron microscope image of a chimeric gas vesicle negatively stained, with a scale bar of 200 nanometers.

[0033] Figure 9 is a flowchart illustrating the preparation process of chimeric gas vesicles.

[0034] Figure 10 is a flowchart illustrating the process of inducing an immune response in individuals after they are given the CGV-Covid-19 vaccine.

Claims

1. A chimeric gas vesicle, comprising: At least two gas vesicle rib proteins, wherein the at least two gas vesicle rib proteins include: a master rib protein comprising GvpB protein derived from P. megaterium; and a primary rib protein comprising: a core sequence comprising amino acid residues 9 to 61 as in SEQ ID NO: 1, the protein sequence of SEQ ID NO: 1 being derived from P. megaterium; and a heteropeptide having at least 6 to 56 amino acids, in frame inserted at the C-terminus of the core sequence; an assembly protein comprising GvpP protein and GvpQ protein; and an accessory protein comprising GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT, and GvpU.

2. The chimeric gas vesicle as described in claim 1, wherein the heteropeptide is derived from a natural peptide sequence selected from a group consisting of ligands, hormones, cytokines, receptors, paratopes of antibodies, toxic proteins, fluorescent proteins, or an artificially designed peptide sequence.

3. The chimeric gas vesicles as described in claim 1, which are derived from and purified from Escherichia coli (E. coli).

4. The chimeric gas vesicle as described in claim 1, wherein the major rib protein and the minor rib protein constitute at least 90% of the dry weight of the chimeric gas vesicle.

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