Adenoviral vector carrying trichinella spiralis CLP gene, construction method therefor, and application thereof

Abstract

An adenoviral vector carrying a trichinella spiralis CLP gene, a construction method therefor, and an application thereof, relating to the technical fields of genetic engineering and recombinant viral vaccines. In order to solve the technical problem of existing trichinella spiralis vaccines being administered as a 2 to 3 dose series and failing to provide long-term efficacy, the present invention uses a trichinella spiralis CLP protein as an antigen, and integrates a gene encoding the CLP protein into an adenovirus vector by means of recombinant DNA technology, so as to obtain a recombinant adenoviral vector; the recombinant adenovirus vector is then transfected into mammalian cells and subjected to packaging and amplification processes to obtain a recombinant adenovirus, with multiple experiments then conducted to confirm the recombinant adenovirus. In addition, animal immunization experiments demonstrate that immunizing mice with recombinant adenovirus rAd5TsCLP achieves effective protection against trichinella spiralis infection. The present invention lays a foundation for subsequent trichinella spiralis vaccine research.

Description

Adenovirus vectors carrying Trichinella spiralis CLP genes, their construction methods and applications Technical Field

[0001] This invention belongs to the field of genetic engineering and recombinant viral vaccine technology, specifically relating to an adenovirus vector carrying the Trichinella spiralis CLP gene, its construction method, and its application. Background Technology

[0002] Trichinosis is a serious zoonotic foodborne parasitic disease caused by the nematode Trichinella spiralis. It is primarily caused by consuming raw or undercooked meat containing Trichinella larval cysts and is widely transmitted among mammals. Trichinosis has a global distribution, with particularly severe outbreaks in Europe and North America. In China, trichinosis is characterized by localized and outbreak-like epidemics, with Yunnan, Tibet, and Henan provinces reporting the most cases. Given the public health risks posed by trichinosis, the European Union has listed it as one of the 15 Important Zoonotic Diseases and a Category A Mandatory Reporting Disease. The World Organisation for Animal Health (WOAH) has listed it as a mandatory disease for slaughtered pigs. my country re-listed it as a key zoonotic disease in 2022. Trichinosis not only poses a serious threat to public health and food safety but also causes significant problems for the animal husbandry industry. Currently, there are no effective preventative measures for trichinosis; therefore, vaccine development has become one of the important means of controlling the disease.

[0003] However, the unique pathogenic biology and immunological characteristics of Trichinella spiralis make vaccine development difficult. This is because, unlike simple pathogens such as bacteria, viruses, protozoa, and fungi, Trichinella spiralis can rapidly trigger a type II immune response (anti-inflammatory / anti-inflammatory response) in the host, suppressing the reactivation of vaccine or infection immune memory, thus leading to vaccine failure or reinfection. The life cycle of Trichinella spiralis is complex. Adult worms primarily parasitize the small intestine, sustaining themselves by ingesting food and producing larvae. These larvae are then transported throughout the body via the lymphatic or bloodstream, but only those reaching the striated muscle can continue to develop. However, before entering the bloodstream and attempting to reach the striated muscle, the larvae must first overcome the intestinal mucosal barrier. The integrity and defense function of the intestinal mucosa are crucial to preventing the invasion of Trichinella spiralis larvae. Once the larvae breach the intestinal mucosal barrier, they can travel through the bloodstream to various organs and tissues throughout the body, causing trichinosis. Therefore, the key to developing a trichinosis vaccine should be to block larval development in the intestine, ensure adult worm survival, and promote the expulsion of adult worms from the intestine.

[0004] With the rapid development of genetic engineering technology, the parallel development of multiple vaccine strategies is an important direction for the research and development of trichinosis vaccines in animals. Currently, animal trichinosis vaccine research can be broadly divided into four types: attenuated vaccines, natural antigen vaccines, recombinant protein vaccines, and DNA vaccines. However, these vaccines require 2-3 doses and cannot achieve long-term effective protection. Therefore, given the time-sensitive nature of animal slaughter, there is an urgent need to develop trichinosis vaccines that can achieve highly effective immunization with a single dose. Viral vector vaccines, as a novel vaccine research strategy, have received widespread attention. In the 1980s, the first viral vector vaccine was successfully developed using recombinant vaccinia virus expressing hepatitis B virus (HBV) surface antigen. Currently, adenovirus vectors, as one of the most widely used viral vectors in vaccine development, can rapidly develop new vaccines by simply altering the delivered nucleic acid sequence. They have advantages such as high transgenic efficiency, high safety, no need for adjuvants, and multiple routes of administration, and have been widely used, such as the recombinant Ebola virus disease vaccine (adenovirus vector) and the recombinant novel coronavirus vaccine (adenovirus type 5 vector) which have obtained approval numbers from the National Medical Products Administration. It is worth noting that by using a human serum-derived type 5 replication-deficient adenovirus vector to integrate the gene of the SARS-CoV-2 S protein into the adenovirus genome, intramuscular and inhaled adenovirus vector-based SARS-CoV-2 vaccines have been successfully developed. This has accumulated more experience for effectively responding to potential future outbreaks of emerging infectious diseases and provided new ideas and strategies for vaccine development in the global public health field. Furthermore, recombinant adenovirus vector vaccines have been applied to animals such as pigs, chickens, and cats, further validating the feasibility of adenovirus vector vaccines as novel vaccines against animal parasites. Summary of the Invention

[0005] To address the technical problem that existing trichinella vaccines require 2-3 doses and lack long-term effectiveness, this invention provides a recombinant adenovirus vector carrying the trichinella cysteine ​​protease inhibitor-like protein (CLP) gene, the recombinant adenovirus, its construction method, and its application in the preparation of a recombinant adenovirus vector vaccine for trichinella disease. The core design of the recombinant adenovirus vector provided by this invention lies in using the CLP protein, which is transcribed at different developmental stages of Trichinella spiralis and reaches its maximum transcription level at 6 hours of intestinal infection with muscle larvae, as an antigen. This protein is integrated into the adenovirus vector using gene recombination technology. The recombinant adenovirus vector obtained in this invention is transfected into HEK293A cells, packaged, amplified, and subjected to repeated freeze-thaw cycles to obtain the recombinant adenovirus. Multiple experiments have confirmed that the recombinant adenovirus rAd5TsCLP expresses the Trichinella spiralis CLP gene and protein in HEK293A cells, with no morphological difference from the wild-type adenovirus Ad5Neg. The target gene insertion is stable and does not affect the biological characteristics of the recombinant adenovirus. Furthermore, this invention has demonstrated through animal immunization experiments that immunizing mice with the recombinant adenovirus rAd5TsCLP effectively resists Trichinella spiralis infection, laying the foundation for subsequent research on Trichinella spiralis vaccines.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention proposes the following technical solution:

[0007] The first objective of this invention is to provide a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene, wherein the recombinant adenovirus vector is obtained by homologous recombination of a replication-defective adenovirus backbone plasmid and a recombinant adenovirus shuttle plasmid carrying the Trichinella spiralis CLP gene; the replication-defective adenovirus backbone plasmid is pAdEasy-1, and the nucleotide sequence of the Trichinella spiralis CLP gene is shown in SEQ ID NO.1.

[0008] The Trichinella spiralis CLP gene, a highly abundant and antigenic gene, was screened from a cDNA library of infective intestinal larvae 6 hours after Trichinella spiralis infection by our research group. It encodes a cysteine ​​protease inhibitor. Cysteine ​​protease inhibitors not only regulate the activity of cysteine ​​proteases such as cathepsin and papain in the parasite, maintaining their physiological homeostasis, but may also participate in the interaction between the parasite and the host, influencing parasite invasion, parasitism, and evasion of the host's immune system. Furthermore, CLP protein inhibits dendritic cell (DC) antigen presentation and hinders T cell activation and differentiation, helping Trichinella spiralis evade the immune response and achieve long-term parasitism. Simultaneously, it inhibits the secretion of pro-inflammatory cytokines (such as IL-6, IL-12, and TNF-α) and promotes the secretion of anti-inflammatory cytokines (such as IL-10), regulating the immune response and reducing damage. CLP protein also inhibits dendritic cell migration, restricts their contact with T cells and B cells, and inhibits the establishment of immune synapses and the initiation of adaptive immune responses.

[0009] A second objective of this invention is to provide a method for constructing the above-mentioned recombinant adenovirus vector, the method comprising the following steps:

[0010] (1) The Trichinella spiralis CLP gene fragment was obtained by amplification using primers with nucleotide sequences as described in SEQ ID NO.2 and SEQ ID NO.3;

[0011] (2) The Trichinella CLP gene fragment obtained in step (1) was ligated into the shuttle plasmid, and the resulting ligation product was introduced into E. coli for amplification and extraction to obtain the recombinant shuttle plasmid.

[0012] (3) The recombinant shuttle plasmid obtained in step (2) is digested with a single enzyme to obtain a linearized recombinant shuttle plasmid. The linearized recombinant shuttle plasmid is transformed into Escherichia coli BJ5183 carrying pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus vector is obtained by screening with kanamycin.

[0013] In one embodiment of the present invention, the shuttle plasmid in step (2) is pShuttle-CMV, and the Escherichia coli is Escherichia coli DH5α.

[0014] A third objective of this invention is to provide an application of the above-mentioned recombinant adenovirus vector, wherein the application is to use the above-mentioned recombinant adenovirus vector to prepare biological products for the prevention and treatment of trichinosis.

[0015] A fourth objective of this invention is to provide a recombinant adenovirus carrying the Trichinella spiralis CLP gene, wherein the recombinant adenovirus is obtained by transfecting mammalian cells with the aforementioned recombinant adenovirus vector.

[0016] In one embodiment of the present invention, the mammalian cell is a HEK293A cell.

[0017] A fifth objective of this invention is to provide a method for preparing the above-mentioned recombinant adenovirus, the method comprising the following steps:

[0018] The recombinant adenovirus vector was linearized and transfected into mammalian cells. The recombinant adenovirus was then packaged and amplified. The diseased cells were collected and subjected to repeated freeze-thaw cycles to obtain the recombinant adenovirus.

[0019] In one embodiment of the present invention, the preparation method is to transfect the recombinant adenovirus vector rAd5TsCLP into HEK293A cells using Lipofectamine 3000, allowing it to recombine and package in HEK293A cells for 14 days, collect cytopathic effector (CPE) cells, repeatedly freeze and thaw the virus and collect it, purify it and freeze it at -80°C.

[0020] The sixth object of the present invention is to provide an application of the above-mentioned recombinant adenovirus, wherein the application is to use the above-mentioned recombinant adenovirus in the preparation of biological products for the prevention and treatment of trichinosis.

[0021] A seventh objective of the present invention is to provide a recombinant adenovirus vector vaccine for trichinosis, wherein the recombinant adenovirus vector vaccine for trichinosis is prepared using the aforementioned recombinant adenovirus as an antigen.

[0022] In one embodiment of the present invention, the immunization method of the trichinosis recombinant adenovirus vector vaccine is intramuscular injection or nasal drop immunization.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene. The core design of this recombinant adenovirus vector utilizes the CLP protein, which is transcribed at different developmental stages of Trichinella spiralis and reaches its maximum transcription level at 6 hours of infecting intestinal myolarvae, as an antigen. This protein is integrated into a replication-defective adenovirus vector using gene recombination technology, resulting in a type 5 replication-defective adenovirus (Ad-5). The obtained recombinant adenovirus vector was transfected into mammalian cells, and after packaging, amplification, and repeated freeze-thaw cycles, recombinant adenovirus was obtained. PCR and Western blot experiments confirmed that the recombinant adenovirus rAd5TsCLP expressed the Ts-CLP gene (Trichinella spiralis CLP gene) and protein in HEK293A cells. Transmission electron microscopy showed that the morphology of the recombinant adenovirus rAd5TsCLP was indistinguishable from that of the wild-type adenovirus Ad5Neg. The growth kinetics curve of the recombinant adenovirus indicated that the target gene insertion was stable and did not affect the biological characteristics of the adenovirus. The above results indicate that recombinant adenovirus rAd5TsCLP has the potential to be used to prepare vaccine products that can induce a specific immune response in the body, laying the foundation for subsequent vaccine research.

[0025] This invention immunized BALB / c mice with recombinant adenovirus rAd5TsCLP via both intramuscular injection and intranasal administration. Two weeks after the initial immunization, the mice were infected with Trichinella spiralis muscle larvae. The differences between intranasal and intramuscular immunization in inducing an immune response and providing protection in BALB / c mice were compared. Both routes induced specific antibodies in BALB / c mice and significantly reduced parasite load. Furthermore, intranasal immunization stimulated a stronger mucosal immune response, inducing high levels of secretory immunoglobulin A (sIgA) in mouse serum and intestinal flushing fluid, and increasing histamine levels in the intestinal flushing fluid. This helps maintain the health and balance of the mucosal immune system, more effectively preventing Trichinella spiralis larvae from invading the intestinal mucosa, interrupting their development, and promoting their excretion. It provides effective protection from the first line of defense against Trichinella spiralis invasion. Therefore, intranasal immunization achieved a slightly higher larval reduction rate compared to intramuscular injection, with a single immunization reducing the muscle larvae count as high as 47.91%. It is evident that using the recombinant adenovirus prepared in this invention for the preparation of a trichinosis vaccine can induce the production of large amounts of specific antibodies such as sIgA in the intestinal mucosa, thereby enhancing the mucosal barrier's defense capabilities and effectively blocking the attachment, invasion, and spread of Trichinella spiralis in the intestine, thus protecting the host from infection. Therefore, this invention provides a new parallel technical pathway for the development of trichinosis vaccines and will also offer valuable reference and inspiration for the development of vaccines against other parasitic diseases. Attached Figure Description

[0026] Figure 1 is a schematic diagram of the construction method of a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene;

[0027] Figure 2 shows the identification results of the target gene Ts-CLP amplification; where M is the DL2000 DNA Marker and 1 is the amplification product.

[0028] Figure 3 shows the identification results of the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP; where M is the DL10000 DNA Marker, and 1 is the recombinant shuttle plasmid pShuttle-CMV and the target gene Ts-CLP after double digestion with HindIII and XhoI.

[0029] Figure 4 shows the linearization effect of PmeI restriction enzyme digestion of recombinant shuttle plasmid pShuttle-CMV-Ts-CLP; where M is DL15000 DNA Marker and 1 is the linearized product of single enzyme digestion of recombinant shuttle plasmid pShuttle-CMV-Ts-CLP using PmeI enzyme.

[0030] Figure 5 shows the PacI restriction enzyme digestion identification results of recombinant adenovirus plasmid rAd5TsCLP; where M is DL15000 DNA Marker, 1 is recombinant adenovirus plasmid rAd5TsCLP, and 2 is the product of PacI enzyme digestion of recombinant adenovirus plasmid rAd5TsCLP.

[0031] Figure 6 shows the pathological changes in HEK293A cells infected with recombinant adenovirus plasmid rAd5TsCLP; where A in Figure 6 represents the group infected with recombinant adenovirus plasmid rAd5TsCLP, and B in Figure 6 represents the negative control group of uninfected cells.

[0032] Figure 7 shows the viral titers of recombinant adenovirus rAd5TsCLP and wild-type adenovirus Ad5Neg as a function of infection time when the multiplicity of infection (MOI) is 0.1; where ns indicates no statistical difference.

[0033] Figure 8 shows the results of PCR verification of the recombinant adenovirus rAd5TsCLP genome;

[0034] Figure 9 shows the results of Western blot analysis of Ts-CLP protein expression in HEK293A cell lysates infected with recombinant adenovirus rAd5TsCLP.

[0035] Figure 10 shows the transmission electron microscopy (TEM) results of recombinant adenovirus rAd5TsCLP; where A in Figure 10 is the TEM result of adenovirus Ad5Neg, and B in Figure 10 is the TEM result of recombinant adenovirus rAd5TsCLP.

[0036] Figure 11 shows the statistical results of the number of muscle larvae and intestinal adult worms in mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; where A in Figure 11 shows the statistical results of the number of muscle larvae in mice, and B in Figure 11 shows the statistical results of the number of intestinal adult worms in mice; ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, and ns indicates no statistical difference;

[0037] Figure 12 shows the detection results of specific antibody levels in the serum of mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; where A in Figure 12 represents the detection results of specific antibody IgG in mouse serum, B represents the detection results of specific antibody IgG1 in mouse serum, C represents the detection results of specific antibody IgG2a in mouse serum, D represents the detection results of specific antibody IgM in mouse serum, and E represents the detection results of specific antibody IgA in mouse serum; * indicates P<0.05, ** indicates P<0.01;

[0038] Figure 13 shows the results of serum neutralizing antibody detection in mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; * indicates P<0.05;

[0039] Figure 14 shows the detection results of total IgA, specific IgA antibody, and histamine levels in the intestinal lavage fluid of mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; where A in Figure 14 shows the detection results of total IgA level in mouse intestinal lavage fluid, B in Figure 14 shows the detection results of specific sIgA antibody in mouse intestinal lavage fluid, and C in Figure 14 shows the detection results of histamine level in mouse intestinal lavage fluid; * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the spirit of the invention will be described in detail below with reference to specific embodiments and accompanying drawings. Any person skilled in the art who understands the embodiments of this invention can make changes and modifications based on the techniques taught in this invention without departing from the spirit and scope of this invention.

[0041] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, enzymes, cells, plasmids, etc. used are all commercially available.

[0043] The adenovirus shuttle plasmid pShuttle-CMV used in this invention was purchased from Beyotime Biotechnology Co., Ltd., with the product name pShuttle-CMV-C-EGFP (adenovirus plasmid, green fluorescence) and product number D8117.

[0044] The adenovirus backbone plasmid pAdEasy-1 used in this invention was purchased from Beyotime Biotechnology Co., Ltd., with the product name pAdEasy-1 / BJ5183 glycerol bacteria (adenovirus recombinant supporting bacteria) and product number D8107.

[0045] The wild-type adenovirus Ad5Neg used in this invention was provided by abm, with a viral titer of 1×10⁻⁶. 10 PFU / mL;

[0046] The BALB / c mice used in this invention were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd., and were SPF grade mice.

[0047] Example 1: Construction method of recombinant adenovirus vector carrying Trichinella spiralis CLP gene

[0048] This invention constructs a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene by integrating the Ts-CLP gene cassette into the E1 region of an Ad5 adenovirus vector with the E1 and E3 regions removed (see Figure 1). The specific construction method is as follows:

[0049] 1. Obtaining the target gene Ts-CLP

[0050] The CDS sequence of Trichinella spiralis cysteine ​​protease inhibitor-like protein (Ts-CLP) was obtained from PubMed (Genebank accession number: EU263325.1). Codon optimization yielded a suitable nucleotide sequence encoding Ts-CLP for expression in mammalian cells, as shown in SEQ ID NO.1, with a full length of 1218 bp. Primers for amplification were designed based on this nucleotide sequence. The upstream primer sequence is shown in SEQ ID NO.2, and the downstream primer sequence is shown in SEQ ID NO.3. The insect cDNA library was amplified using these primers (PCR reaction program: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min 10 s, 35 cycles; 72℃ extension for 10 min). The amplified products were obtained and identified by agarose gel electrophoresis. The identification results are shown in Figure 2. As shown in Figure 2, the fragment size of the amplified product is approximately 1218 bp, proving that the target gene fragment was successfully obtained.

[0051] SEQ ID NO.1:

[0052] SEQ ID NO.2: 5′-AAGCTTATGAGCTTCATGCACTGC-3′ (the italicized part is the HindIII restriction site);

[0053] SEQ ID NO.3: 5′-CTCGAGTTAGCACTCCACGGTGC-3′ (the italicized part is the XhoI restriction site).

[0054] 2. Construction of a recombinant adenovirus vector expressing the Ts-CLP gene

[0055] A recombinant adenovirus vector expressing the Ts-CLP gene was constructed using the E1 and E3-deficient adenovirus type 5 AdEasy-1 system. The specific steps are as follows:

[0056] The target gene Ts-CLP was cloned into the multiple cloning site region of the pShuttle-CMV plasmid using HindIII and XhoI restriction sites, constructing the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP carrying the target gene. The recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was identified by double digestion with HindIII and XhoI, and the results are shown in Figure 3. Figure 3 shows that the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was successfully constructed.

[0057] The constructed recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was transformed into E. coli DH5α competent cells for amplification. Single clones were picked and cultured overnight, and the plasmid was extracted using a small-scale extraction kit. The extracted recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was linearized by digestion with PmeI, and the linearization effect was confirmed by nucleic acid electrophoresis (see Figure 4).

[0058] The linearized recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was transformed into BJ5183 bacteria already carrying the pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus plasmid in BJ5183 bacteria was kanamycin resistant; therefore, the transformed bacteria were inoculated onto kanamycin-resistant plates for culture. After overnight culture, clones of varying sizes were observed, while the recombinant adenovirus plasmid, being large and growing slowly, typically resulted in smaller clones. Smaller clones were picked and inoculated into LB broth containing kanamycin and cultured overnight. A small amount of the obtained bacterial culture was used to extract the plasmid, which was then digested with PacI, and the digestion effect was assessed (see Figure 5). As shown in Figure 5, PacI digestion of the recombinant adenovirus plasmid yielded a large fragment of approximately 30 kb and a smaller fragment of 3 kb or 4.5 kb.

[0059] The correctly identified recombinant adenovirus plasmid rAd5TsCLP was transformed into XL10-Gold supercompetent bacteria for amplification. The amplified recombinant adenovirus plasmid was digested with PacI, and a small amount of the digested plasmid was used for electrophoresis identification. The remaining digested products were purified into DNA and used for subsequent adenovirus packaging.

[0060] Example 2: Preparation method of recombinant adenovirus carrying Trichinella spiralis CLP gene

[0061] 1. Packaging of recombinant adenovirus rAd5TsCLP

[0062] 7×10 5 -8×10 5 HEK293A cells were cultured in 60 mm cell culture dishes at a concentration of 1 / mL and cultured for 24 hours. The recombinant adenovirus plasmid obtained in Example 1, digested with PacI, was transfected into the overnight adherent HEK293A cells using Lipofectamine 3000. Six hours after transfection, the culture medium was replaced with fresh medium and cultured for another 7-10 days. During this period, the color of the culture medium should be monitored; if it turns yellow, the medium should be changed. When changing the medium, a portion of the culture medium should be carefully aspirated, and then an appropriate amount of fresh culture medium should be added. After 7-10 days of culture, if a large number of empty plaques are observed in the cells in the culture dish, or if the cells are clearly floating (see Figure 6), the adenovirus can be collected.

[0063] 2. Collection and amplification of recombinant adenovirus rAd5TsCLP

[0064] Carefully aspirate the cell culture medium obtained from the adenovirus packaging in step 1, avoiding contact with the cells. Add 0.5 mL of PBS, scrape off the cells, and transfer them to a 1.5 mL centrifuge tube. Lyse the cells using a repeated freeze-thaw method, specifically by repeatedly freezing and thawing at -80°C and 37°C four times. After freeze-thaw, centrifuge and collect the supernatant, which is the P0 generation adenovirus, for subsequent amplification and purification. Aliquot and store at -80°C. Infect HEK293A cells with the P0 generation adenovirus to amplify the virus. Specifically, add the P0 generation virus to fresh cell culture medium for small-scale amplification until plaques appear in the cells. Collect and repeatedly freeze and thaw four times to obtain the P1 generation virus. Infect three consecutive generations up to the P4 generation for large-scale amplification. After plaque formation, collect, purify, and concentrate the virus. After titer determination, aliquot the recombinant adenovirus rAd5TsCLP and store at -80°C.

[0065] 3. Identification of recombinant adenovirus rAd5TsCLP

[0066] (1) Titer determination of recombinant adenovirus

[0067] With a concentration of 1×10 5 HEK293A cells were seeded at / mL in 96-well plates and cultured for 24 hours. The recombinant adenovirus rAd5TsCLP solution was then serially diluted 10-fold and added to 96-well plates containing HEK293A cells, with eight parallel wells for each dilution. Wild-type adenovirus Ad5Neg was also seeded into HEK293A cells using the same method. A blank control was also included. Cells were cultured for another 96 hours after infection, during which cytopathic effects and fluorescence expression were observed. The viral titer at different time points was calculated using the Reed-Muench method. The titer results are shown in Figure 7. Figure 7 shows that after 96 hours of infection, there was no significant difference in viral titer between recombinant adenovirus rAd5TsCLP and wild-type adenovirus Ad5Neg, and their growth trends were consistent, indicating that the insertion of the exogenous protein did not affect the growth performance of the recombinant adenovirus.

[0068] (2) Recombinant adenovirus genome PCR identification

[0069] The recombinant adenovirus rAd5TsCLP genome was extracted from the supernatant of infected cells using a viral gene extraction kit, following the kit's instructions. The recombinant adenovirus genome was then validated by PCR. The results showed that the amplified product was 1218 bp in size, consistent with the expected size of the target gene (Ts-CLP gene) (see Figure 8).

[0070] (3) Western blot analysis of recombinant adenovirus Ts-CLP protein expression

[0071] Supernatant and lysis buffer from virus-packing cells were collected (lysis working buffer was prepared by adding 10 μL of 100 mM PMSF to 1 mL of RIPA lysis buffer). Protein samples were separated using SDS-PAGE. A methanol-activated PVDF membrane was then attached to the gel and transferred at 220 mA for 2 h. The PVDF membrane was blocked in TBST buffer containing 5% skim milk for 1 h, and then incubated overnight at 4°C with anti-Ts-CLP mouse monoclonal antibody (1:2000 dilution). After washing three times with TBST, the membrane was incubated with goat anti-mouse IgG (H+L)HRP antibody at room temperature for 2 h, followed by five TBST washes. The results were analyzed using a UVP Chemstudio instrument. The results showed that the Ts-CLP protein size was 45.9 kDa, consistent with the expected target band size (see Figure 9).

[0072] (4) Morphological observation by transmission electron microscopy

[0073] Viral droplets of adenovirus Ad5Neg and recombinant adenovirus rAd5TsCLP were placed on a 200-mesh electron microscope screen (copper mesh), stained with 1% phosphotungstic acid for 1 min, and excess liquid was aspirated. The particles of both adenoviruses were observed using a transmission electron microscope. The results showed that the recombinant adenovirus rAd5TsCLP exhibited a regular icosahedral morphology, similar to the particles of the empty vector Ad5Neg virus. This indicates that after the insertion of the exogenous target gene, the adenovirus can undergo normal packaging and maintain its natural morphology (see Figure 10).

[0074] Example 3: Application of recombinant adenovirus carrying the Trichinella spiralis CLP gene in vaccine preparation

[0075] To determine whether the recombinant adenovirus carrying the Trichinella spiralis CLP gene obtained in this invention can be used to prepare a Trichinella spiralis vaccine, the immunogenicity of the recombinant adenovirus in BALB / c mice was evaluated. The specific method is as follows:

[0076] This invention employs two methods of immunization: intramuscular injection (IM) and intranasal administration (IN). Mice were immunized with recombinant adenovirus rAd5TsCLP. To evaluate the immunization effect of rAd5TsCLP on mice, the number of adult intestinal worms 3 days after oral infection with Trichinella spiralis muscle larvae and the number of muscle larvae 5 weeks after infection were measured (expressed as LPG of larvae per gram of muscle), and the worm reduction rate was calculated. Simultaneously, blood samples were collected at 0, 2, and 7 weeks after the initial immunization to detect the levels of specific antibodies (IgG, IgG1, IgG2a, IgM, IgA) and neutralizing antibody NAb in mouse serum, as well as the levels of total sIgA, specific sIgA, and histamine in intestinal lavage fluid.

[0077] 1. Immunized animals

[0078] 10 were administered via both intramuscular injection and nasal drops. 8 PFU recombinant adenovirus rAd5TsCLP was administered to 6-8 week old BALB / c mice (10 mice per group). A treatment receiving adenovirus Ad5Neg served as a vaccine control, and a treatment receiving PBS served as a blank control. To evaluate the immunization effect, two weeks after the initial immunization, mice were orally infected with 250 Trichinella spiralis muscle larvae per mouse.

[0079] 2. Detection of the number of muscle larvae and intestinal adult worms in mice

[0080] Mice were euthanized on day 3 and week 5 post-infection to determine the number of adult intestinal worms and muscle larvae, and to calculate the reduction rate. The number of muscle larvae was determined using a pepsin digestion method, as follows: pepsin and hydrochloric acid were added to distilled water at 37°C to prepare a digestion solution. The final concentrations of both pepsin and hydrochloric acid were 1%. Each mouse was digested with 500 mL of the digestion solution and placed in a 37°C incubator with a magnetic stirrer for 2 hours. After the entire digestion process, the residue in the digestion solution was filtered out through a 60-mesh sieve. The solution was allowed to stand at room temperature for 1 hour, and the supernatant was gently aspirated with a sterile syringe. 100 mL of the liquid was collected, and 400 mL of distilled water was added. The mixture was washed repeatedly, and finally, the liquid was transferred to a 50 mL beaker for microscopic counting of the Trichinella spiralis muscle larvae.

[0081] The method for calculating the reduction rate of muscle larvae is as follows:

[0082] Based on the number of muscle larvae in each mouse, the number of muscle larvae per gram of muscle (LPG) in each mouse was calculated. The average LPG of each group of mice was calculated and compared with the LPG of the PBS control group to calculate the larvae reduction rate of each experimental group. The formula is as follows: Muscle larvae reduction rate (%) = (1 - average LPG of experimental group / LPG of PBS control group) × 100%.

[0083] The test results are shown in Figure 11. Five weeks after infection, compared with the PBS group, the myolar worm load in the IM group, IN group, and Ad5Neg empty vector group decreased by 45.61%, 47.91%, and 10.57%, respectively. This indicates that different methods of inoculation with the recombinant virus rAd5TsCLP all resulted in worm reduction in mice and provided a protective effect (see Figure 11, A). Furthermore, compared with the PBS group, immunization with the IM group, IN group, and Ad5Neg empty vector group reduced the adult worm load on day three after Trichinella spiralis infection by 36.49%, 42.57%, and 10.59%, respectively. Therefore, for the intestinal stage, nasal drop immunization provided better protection than intramuscular injection, and there was no significant difference between the PBS group and the Ad5Neg group (see Figure 11, B).

[0084] 3. Detection of specific antibody levels in mouse blood

[0085] Specific antibodies (IgG, IgG1, IgG2a, IgM, IgA) in the serum of mice in each group were detected by indirect enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: 5 μg / mL of purified recombinant protein rTs-CLP was coated onto 96-well plates and incubated overnight at 4°C; then 100 μL / well blocking buffer (5% skim milk powder) was added, and the plates were blocked at 37°C for 1 h; the collected serum was serially diluted 10-fold and incubated at 37°C for 1 h; goat anti-mouse IgG-HRP was added for incubation; finally, TMB substrate was added for color development, and the reaction was terminated with 2M H2SO4 sulfuric acid. The OD was measured using a microplate reader. 450 value.

[0086] The results showed that, two weeks after the initial immunization, the IgG antibody titers in the IM and IN groups showed a significant increasing trend compared to the PBS and Ad5Neg groups. The serum samples were diluted 50-fold to detect the levels of IgG1, IgG2a, IgM, and IgA specific antibodies. No significant differences were found between the PBS and Ad5Neg groups at any of the detection time points. Five weeks after challenge (seven weeks after immunization), the levels of anti-Ts-CLP IgG1, IgG2a, IgM, and IgA antibodies in both control groups increased. Compared to the control groups, the IM group showed significantly higher levels of IgG1, IgG2a, IgM, and IgA antibodies. The IN group also induced antibody levels comparable to the IM group, and compared to the IM group, the IN group induced higher titers of mucosal immunity-associated IgA antibodies (see Figure 12).

[0087] 4. Detection of neutralizing antibodies in mouse blood

[0088] The collected serum was inactivated at 56°C for 30 min, and then serially diluted 2-fold in 96-well plates (1:10, 1:20, 1:40, 1:80, 1:160, and 1:320 in 100 μL of DMEM), and then mixed with an equal volume of 100 TCID45. 50 Recombinant adenovirus rAd5TsCLP was mixed and incubated at 37°C for 1 hour. Then, 200 μL of the serum-virus mixture was added to a monolayer of HEK293A cells. HEK293A cells treated with recombinant adenovirus Ad5Neg and uninoculated cells served as control groups. Cells were cultured at 37°C and 5% CO2 for 72 hours, and the expression of viral fluorescent proteins was observed. Neutralizing antibody titers were calculated. A positive result for neutralizing antibodies was defined as a reduction in the number of green fluorescent lesions in the diluted sample compared to the infected control group by more than 50%. The neutralizing antibody titers against rAd5TsCLP in both the IM and IN groups showed a significant increasing trend 2 weeks after immunization (see Figure 13).

[0089] 5. Analysis of sIgA and histamine levels in mouse intestines

[0090] After euthanasia of mice, the small intestine was removed and repeatedly flushed (intestinal flushing fluid: PBS buffer containing 1% EDTA and PMSF). The intestinal flushing fluid was collected into EP tubes, centrifuged at 4°C for 5 min, and the supernatant was separated. Total sIgA and specific sIgA of Trichinella spiralis were detected using an indirect ELISA method. The specific method is as follows: crude Trichinella spiralis protein and Ts-CLP protein were coated onto an ELISA plate, with intestinal flushing fluid as the primary antibody and goat anti-mouse IgA as the secondary antibody. The OD value was measured at 450 nm. Histamine levels in the intestinal fluid were measured at 0, 2, and 7 weeks post-immunization, and histamine concentration in the intestine was detected using a mouse ELISA kit. The results showed that total sIgA and Ts-CLP-specific sIgA antibody levels in the intestinal flushing fluid were detectable for five weeks post-challenge (7 weeks post-immunization). Furthermore, the IN group showed higher levels of total sIgA and Ts-CLP-specific sIgA compared to the IM group. The IN group showed higher histamine levels compared to the IM group. Five weeks after infection (seven weeks after immunization), histamine levels returned to the normal levels of the control group. The IN group induced a more pronounced intestinal mucosal reaction and histamine secretion level compared to the IM group (see Figure 14).

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant adenovirus vector carrying the Trichinella spiralis CLP gene, characterized in that, The recombinant adenovirus vector was obtained by homologous recombination of a replication-defective adenovirus backbone plasmid and a recombinant adenovirus shuttle plasmid carrying the Trichinella spiralis CLP gene; the replication-defective adenovirus backbone plasmid is pAdEasy-1, and the nucleotide sequence of the Trichinella spiralis CLP gene is shown in SEQ ID NO.

1.

2. The method for constructing the recombinant adenovirus vector according to claim 1, characterized in that, Includes the following steps: (1) The Trichinella spiralis CLP gene fragment was obtained by amplification using primers with nucleotide sequences as described in SEQ ID NO.2 and SEQ ID NO.3; (2) The Trichinella CLP gene fragment obtained in step (1) was ligated into the shuttle plasmid, and the resulting ligation product was introduced into E. coli for amplification and extraction to obtain the recombinant shuttle plasmid. (3) The recombinant shuttle plasmid obtained in step (2) is digested with a single enzyme to obtain a linearized recombinant shuttle plasmid. The linearized recombinant shuttle plasmid is transformed into Escherichia coli BJ5183 carrying pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus vector is obtained by screening with kanamycin.

3. The construction method according to claim 2, characterized in that, The shuttle plasmid in step (2) is pShuttle-CMV, and the Escherichia coli is Escherichia coli DH5α.

4. The application of the recombinant adenovirus vector according to claim 1, characterized in that, The application is to use the recombinant adenovirus vector of claim 1 to prepare biological products for the prevention and treatment of trichinosis.

5. A recombinant adenovirus carrying the Trichinella spiralis CLP gene, characterized in that, The recombinant adenovirus was obtained by transfecting mammalian cells with the recombinant adenovirus vector of claim 1.

6. The recombinant adenovirus according to claim 5, characterized in that, The mammalian cells in question are HEK293A cells.

7. The method for preparing the recombinant adenovirus according to any one of claims 5 or 6, characterized in that, The procedure includes the following steps: linearizing the recombinant adenovirus vector as described in claim 1 and transfecting it into mammalian cells, packaging and amplifying the recombinant adenovirus, collecting the diseased cells and obtaining the recombinant adenovirus through repeated freeze-thaw cycles.

8. The application of the recombinant adenovirus according to claim 5 or 6, characterized in that, The application is to use the recombinant adenovirus of claim 5 or 6 to prepare biological products for the prevention and treatment of trichinosis.

9. A recombinant adenovirus vector vaccine for trichinosis, characterized in that, The trichinosis recombinant adenovirus vector vaccine is prepared using the recombinant adenovirus as described in claim 5 or 6 as the antigen.

10. The trichinosis recombinant adenovirus vector vaccine according to claim 9, characterized in that, The recombinant adenovirus vector vaccine for trichinosis is administered via intramuscular injection or nasal drops.

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