Adenovirus vector carrying Trichinella spiralis CLP gene and its construction method and application

Abstract

The invention relates to an adenovirus vector carrying the CLP gene of Trichinella spiralis and a construction method and application thereof, belonging to the technical field of genetic engineering and recombinant virus vaccines. In order to solve the technical problem that the existing Trichinella spiralis vaccine needs to be vaccinated for 2-3 doses and does not have long-term effectiveness, the present invention uses the Trichinella spiralis CLP protein as an antigen, integrates the gene encoding the CLP protein into the adenovirus vector by gene recombination technology, and obtains a recombinant adenovirus vector; then the recombinant adenovirus vector is transfected into mammalian cells, and a recombinant adenovirus is obtained through packaging, amplification and other processes, and the recombinant adenovirus is identified through multiple experiments; in addition, the present invention also proves through immune animal experiments that the use of recombinant adenovirus rAd5TsCLP to immunize mice can achieve the effect of effectively resisting Trichinella spiralis infection. The present invention lays a foundation for the subsequent research on trichinellosis vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and recombinant virus vaccines, and specifically relates to an adenovirus vector carrying a Trichinella spiralis CLP gene and a construction method and application thereof. Background Art

[0002] Trichinellosis is a serious zoonotic foodborne parasitic disease caused by the nematode Trichinella spiralis. It is mainly caused by eating raw or half-cooked meat containing Trichinella larvae cysts and is widely spread among mammals. Trichinellosis is widely distributed worldwide, especially in Europe and North America. In China, trichinellosis is characterized by localized and explosive infection epidemics, with more reports of cases in Yunnan, Tibet and Henan. In view of the harm of trichinellosis to public health, the European Union lists it as one of the 15 important zoonotic diseases and a Class A mandatory reportable disease, and the World Organization for Animal Health (WOAH) lists it as a mandatory disease for slaughtering pigs. In 2022, my country once again listed it as a key disease in the catalog of zoonotic infectious diseases. Trichinellosis not only poses a serious threat to public health and food safety, but also brings serious problems to the animal husbandry industry. At present, there is a lack of effective prevention measures for trichinellosis. Therefore, vaccine research and development has become one of the important means of preventing and controlling trichinellosis.

[0003] However, the unique pathogenic biology and immunological characteristics of Trichinella have made vaccine development difficult. The reason is that Trichinella is different from simple pathogens such as bacteria, viruses, protozoa and fungi. It can quickly trigger the host type II immune response (anti-inflammatory / anti-inflammatory response), inhibit the awakening of vaccine or infection immune memory, and thus lead to host vaccine immunity failure or reinfection. The life history of Trichinella is complex. Adults mainly parasitize in the small intestine, maintain life by ingesting food, and produce larvae. These larvae are then carried throughout the body through lymphatic or blood circulation, but only larvae that reach striated muscle can continue to develop. However, before the larvae enter the blood circulation and try to reach the striated muscle, they must first break through the barrier of the intestinal mucosa. The integrity and defense function of the intestinal mucosa are crucial to preventing the invasion of Trichinella larvae. Once the larvae break through the intestinal mucosal barrier, they can flow with the blood to various organs and tissues throughout the body, thereby causing trichinosis. Therefore, the key points of the development of vaccines for trichinosis should be aimed at blocking the development of larvae in the intestine, the survival of adults, and promoting the excretion of adults in the intestine.

[0004] With the rapid development of genetic engineering technology, the parallel development of multi-type vaccine strategies is an important direction for the development of animal trichinellosis vaccines. At present, animal trichinellosis vaccine research can be roughly 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, in view of the timeliness requirements for animal marketing, it is urgent to develop a trichinellosis vaccine that can achieve high immunity levels with one dose. Viral vector vaccines have attracted widespread attention as a new vaccine research strategy. In the 1980s, the first viral vector vaccine was successfully developed using recombinant vaccinia virus expressing hepatitis B virus (HBV) surface antigen. At present, adenovirus vectors are one of the most widely used viral vectors in vaccine development. New vaccines can be quickly developed by simply changing the delivered nucleic acid sequence. They have the advantages of high transgenic efficiency, high safety, no adjuvant, and can be vaccinated in multiple ways. They have been widely used, such as the recombinant Ebola virus disease vaccine (adenovirus vector) and the recombinant new coronavirus vaccine (adenovirus type 5 vector) approved by the National Medical Products Administration. It is worth noting that the gene of the new coronavirus (SARS-CoV-2) S protein was integrated into the adenovirus genome using a human serum type 5 replication-deficient adenovirus vector, and intramuscular and inhaled adenovirus vector new coronavirus vaccines were successfully developed, which has accumulated more experience for effectively responding to new infectious diseases that may appear in the future, and also provided new ideas and strategies for vaccine development in the field of global public health. In addition, recombinant adenovirus vector vaccines have been used in pigs, chickens, cats and other animals, which further verifies the feasibility of adenovirus vector vaccines as new animal parasite vaccines. Summary of the invention

[0005] In order to solve the technical problem that the existing Trichinella vaccine requires 2-3 doses and has no long-term effectiveness, the present invention provides a recombinant adenovirus vector carrying the Trichinella cysteine ​​protease inhibitor-like protein (CLP) gene, a recombinant adenovirus and a construction method thereof, and its use in preparing a recombinant adenovirus vector vaccine for trichinellosis. The core of the design of the recombinant adenovirus vector provided by the present invention is to use the CLP protein that is transcribed at different developmental stages of Trichinella spiralis and whose transcription level reaches the maximum at 6-hour intestinal infectious muscle larvae as an antigen, and integrate it into the adenovirus vector through gene recombination technology; the present invention transfects the obtained recombinant adenovirus vector into HEK293A cells, and obtains the recombinant adenovirus through packaging, amplification and repeated freezing and thawing, and confirms through multiple experiments that the recombinant adenovirus rAd5TsCLP expresses the Trichinella spiralis CLP gene and protein in HEK293A cells, and its morphology is no different from that of the wild adenovirus Ad5Neg, the insertion of the target gene is stable, and the biological characteristics of the recombinant adenovirus are not affected; in addition, the present invention also proves through animal immunization experiments that the use of the recombinant adenovirus rAd5TsCLP to immunize mice has an effective effect of resisting Trichinella spiralis infection, laying a foundation for the subsequent research on trichinellosis vaccines.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention proposes the following technical solutions:

[0007] The first object of the present 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 is a highly abundant and antigenic gene obtained by our research group from the cDNA library of intestinal infectious larvae 6 hours after Trichinella spiralis infection. It encodes a cysteine ​​protease inhibitor. Cysteine ​​protease inhibitors can not only regulate the activity of cysteine ​​proteases such as cathepsins and papain in the parasite to maintain its physiological balance, but also may participate in the interaction between the parasite and the host, affecting the invasion, parasitism and escape from the host's immune system. In addition, CLP protein inhibits antigen presentation by dendritic cells (DCs), hinders the activation and differentiation of T cells, and helps Trichinella spiralis escape from immune responses and achieve long-term parasitism. At the same time, it inhibits the secretion of pro-inflammatory cytokines (such as IL-6, IL-12, TNF-α) by immune cells, promotes the secretion of anti-inflammatory cytokines (such as IL-10), regulates immune responses, and reduces damage. CLP protein also inhibits the migration of dendritic cells, limits their contact with T cells and B cells, and inhibits the establishment of immune synapses and the initiation of acquired immune responses.

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

[0010] (1) using primers with nucleotide sequences such as SEQ ID NO.2 and SEQ ID NO.3 to amplify and obtain a Trichinella spiralis CLP gene fragment;

[0011] (2) ligating the Trichinella spiralis CLP gene fragment obtained by amplification in step (1) into a shuttle plasmid, and introducing the obtained ligation product into Escherichia coli for amplification, and extracting and obtaining a recombinant shuttle plasmid;

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

[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] The third object of the present 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 for preparing a biological product for preventing and treating trichinosis.

[0015] The fourth object of the present 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 above-mentioned recombinant adenovirus vector.

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

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

[0018] The above-mentioned recombinant adenovirus vector is linearized and then transfected into mammalian cells, and the recombinant adenovirus is packaged and amplified. The diseased cells are collected and repeatedly frozen and thawed 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 by Lipofectamine 3000, so that it is recombined and packaged in HEK293A cells for 14 days, collect cytopathic effect (CPE) cells, repeatedly freeze and thaw to collect the virus, and freeze and store at -80°C after purification.

[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 to prepare a biological product for preventing and treating trichinosis.

[0021] The seventh object of the present invention is to provide a recombinant adenovirus vector vaccine for trichinellosis, which is prepared using the above-mentioned recombinant adenovirus as an antigen.

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

[0023] Beneficial effects of the present invention:

[0024] The present invention provides a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene. The core of the design of the recombinant adenovirus vector is to use the CLP protein that is transcribed at different developmental stages of Trichinella spiralis and reaches the maximum transcription level at 6 h intestinal infective muscle larvae as an antigen, and integrate it into a replication defective adenovirus vector through gene recombination technology to obtain a type 5 replication defective recombinant adenovirus (5 type replication defective adenovirus, Ad-5). The present invention transfects the obtained recombinant adenovirus vector into mammalian cells, and obtains the recombinant adenovirus through packaging, amplification and repeated freezing and thawing. PCR and Western Blot experiments confirm that the recombinant adenovirus rAd5TsCLP expresses the Ts-CLP gene (Trichinella spiralis CLP gene) and protein in HEK293A cells; transmission electron microscopy observation shows that the morphology of the recombinant adenovirus rAd5TsCLP is no different from that of the wild adenovirus Ad5Neg; the growth kinetic curve of the recombinant adenovirus shows that the insertion of the target gene is stable and does not affect the biological characteristics of the adenovirus. The above results indicate that the recombinant adenovirus rAd5TsCLP has the potential to be used to prepare vaccine products that can induce specific immune responses in the body, laying the foundation for subsequent vaccine research.

[0025] The present invention uses intramuscular injection and intranasal immunization to inoculate BALB / c mice with recombinant adenovirus rAd5TsCLP, and infects the mice with Trichinella muscle larvae two weeks after the first immunization. The differences between the two different vaccination routes of intranasal immunization and intramuscular injection in inducing immune response and providing protection in BALB / c mice are compared and evaluated. It is found that both intranasal immunization and intramuscular injection can induce BALB / c mice to produce specific antibodies and significantly reduce the parasite load. In addition, intranasal immunization can stimulate a stronger mucosal immune level, induce a high level of secretory immunoglobulin A (sIgA) in mouse serum and intestinal lavage fluid, increase the histamine content in intestinal lavage fluid, thereby helping to maintain the health and balance of the mucosal immune system, more effectively prevent Trichinella larvae from invading the intestinal mucosa, interrupt their development, and promote excretion, providing effective protection from the first line of defense against Trichinella invasion. Therefore, intranasal immunization achieves a slightly higher worm reduction rate than intramuscular injection immunization, and the muscle larvae reduction rate after a single immunization is as high as 47.91%. It can be seen that the recombinant adenovirus prepared by the present invention is used for the preparation of a trichinosis vaccine, and the obtained vaccine can induce the intestinal mucosa to produce a large amount of specific antibodies such as sIgA, enhance the defense ability of the mucosal barrier, and effectively block the attachment, invasion and spread of Trichinella in the intestine, thereby protecting the host from infection. Therefore, the present invention provides a new parallel technical path for the development of trichinosis vaccines, and will also provide useful references and inspirations for the development of vaccines for other parasitic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 3 This is the identification result of the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP; where M is DL10000 DNAMarker, 1 is Hind III and XOt I recombinant shuttle plasmid pShuttle-CMV and target gene Ts-CLP after double restriction digestion;

[0029] Figure 4 For the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP Pme I enzyme linearization effect diagram; M is DL15000 DNA Marker, 1 is the use of Pme The linearized product of the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP digested by enzyme I;

[0030] Figure 5 For the recombinant adenovirus plasmid rAd5TsCLP Pac I enzyme digestion identification result diagram; wherein M is DL15000 DNAMarker, 1 is the recombinant adenovirus plasmid rAd5TsCLP, 2 is the use of Pac The product of enzyme I digestion of the recombinant adenovirus plasmid rAd5TsCLP;

[0031] Figure 6 This is the pathological picture of HEK293A cells infected with the recombinant adenovirus plasmid rAd5TsCLP; Figure 6 A in the figure is the recombinant adenovirus plasmid rAd5TsCLP infection group, Figure 6 B in the figure is the negative control group of uninfected cells;

[0032] Figure 7 It is a curve diagram showing the changes of virus titers of recombinant adenovirus rAd5TsCLP and wild-type adenovirus Ad5Neg with infection time when the multiplicity of infection MOI is 0.1; wherein ns indicates no statistical difference;

[0033] Figure 8 This is a diagram showing the results of using PCR to verify the genome of the recombinant adenovirus rAd5TsCLP;

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

[0035] Fig.10 Figure 1 is a transmission electron microscopy observation result of the recombinant adenovirus rAd5TsCLP; Fig.10 A in the figure is the transmission electron microscope observation result of adenovirus Ad5Neg. Fig.10 B in the figure is the transmission electron microscopy observation result of the recombinant adenovirus rAd5TsCLP;

[0036] Fig.11 This is a statistical diagram of the number of muscle larvae and intestinal adult worms in mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; Fig.11 A in the figure is the statistical result of the number of larvae in mouse muscle. Fig.11 B in the figure is the statistical result of the number of adult worms in the mouse intestine; ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference;

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

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

[0039] Fig.14 The figure shows the results of the detection of total IgA, specific IgA antibodies and histamine levels in the intestinal lavage fluid of mice immunized with recombinant adenovirus rAd5TsCLP after infection with Trichinella spiralis muscle larvae; Fig.14 A in the figure is the test result of total IgA level in mouse intestinal lavage fluid. Fig.14 B in the figure is the detection result of specific sIgA antibodies in the intestinal lavage fluid of mice. Fig.14 C in the figure is the detection result of histamine level in the intestinal lavage fluid of mice; * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention by the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0041] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

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

[0043] The adenovirus shuttle plasmid pShuttle-CMV used in the present invention was purchased from Bio-Tech Biotech Co., Ltd., the product name is pShuttle-CMV-C-EGFP (adenovirus plasmid, green fluorescence), the product number is D8117;

[0044] The adenovirus backbone plasmid pAdEasy-1 used in the present invention was purchased from Bio-Technology Co., Ltd., the product name is pAdEasy-1 / BJ5183 glycerol bacteria (adenovirus recombinant supporting bacteria), and the product number is D8107;

[0045] The wild-type adenovirus Ad5Neg used in the present invention was provided by abm, and the virus titer was 1×10 10 PFU / mL;

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

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

[0048] The present invention constructs a recombinant adenovirus vector carrying the Trichinella spiralis CLP gene by integrating the Ts-CLP gene cassette into the E1 region of the 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 the Trichinella spiralis cysteine ​​protease inhibitor-like protein (Ts-CLP) was obtained from PubMed (Genebank accession number: EU263325.1). After codon optimization, a nucleotide sequence encoding Ts-CLP suitable for expression in mammalian cells was obtained, as shown in SEQ ID NO.1, with a total length of 1218 bp. Primers for amplification were designed based on the above nucleotide sequence, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3. The above primers were used to amplify the insect cDNA library (PCR reaction program: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 1 min 10s, 35 cycles; 72℃ extension for 10 min), and the amplified product was obtained. The amplified product was identified by agarose gel electrophoresis. The identification results are shown in Figure 2 .Depend on Figure 2 It can be seen that the fragment size of the amplified product is about 1218 bp, proving that the target gene fragment was successfully obtained.

[0051] SEQ ID NO.1:

[0052]

[0053] SEQ ID NO.2:5′- AAGCTT ATGAGCTTCATGCACTGC-3′ (the italic part is Hind III restriction site);

[0054] SEQ ID NO.3:5′- CTCGAG TTAGCACTCCACGGTGC-3′ (the italic part is XOt I restriction site).

[0055] 2. Construction of recombinant adenovirus vector expressing Ts-CLP gene

[0056] The 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:

[0057] use Hind III and XOt I restriction site, clone the target gene Ts-CLP into the multiple cloning site region of the pShuttle-CMV plasmid, and construct the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP carrying the target gene. Hind III and XOt I double enzyme digestion identification, the results are shown in Figure 3 ,Depend on Figure 3 It can be seen that the recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was successfully constructed.

[0058] The constructed recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was transformed into E. coli DH5α competent cells for amplification, a single clone was selected and cultured overnight, and the plasmid was extracted using a small amount extraction kit. Pme I enzyme linearization, and nucleic acid electrophoresis to confirm the plasmid linearization effect (see Figure 4 ).

[0059] The linearized recombinant shuttle plasmid pShuttle-CMV-Ts-CLP was transformed into BJ5183 bacteria that already carried the pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus plasmid in the BJ5183 bacteria is resistant to kanamycin, so the transformed bacteria were inoculated on kanamycin-resistant plates for culture. After overnight culture, it can be seen that the clones are of different sizes, and the recombinant adenovirus plasmid is large in size and grows slowly, and the clones formed are usually small. Pick a smaller clone and inoculate it into LB liquid culture medium containing kanamycin and culture it overnight. A small amount of the obtained bacterial liquid is used to extract the plasmid, and then use Pac I was used for enzyme digestion and the digestion effect was identified (see Figure 5 ).Depend on Figure 5 It can be seen that the recombinant adenovirus plasmid Pac After digestion with enzyme I, a large fragment of about 30 Kb and a small fragment of 3 Kb or 4.5 Kb were obtained.

[0060] The correctly identified recombinant adenovirus plasmid rAd5TsCLP was transformed into XL10-Gold super competent bacteria for amplification. Pac I enzyme digestion, a small amount of the digested plasmid was used for electrophoresis identification, and the remaining digested product was used for subsequent adenovirus packaging after DNA purification.

[0061] Example 2: Preparation of a recombinant adenovirus carrying the Trichinella spiralis CLP gene

[0062] 1. Packaging of recombinant adenovirus rAd5TsCLP

[0063] Inoculate 7×10 5 -8×10 5 / mL HEK293A cells were placed in a 60 mm cell culture dish and cultured for 24 hours. Pac The recombinant adenovirus plasmid after I digestion was transfected into HEK293A cells that were cultured overnight and adherent to the wall by Lipofectamine 3000. Six hours after transfection, fresh cell culture medium was replaced and cultured for 7-10 days. During this period, the color of the culture medium should be paid attention to. If it turns yellow, the medium should be replaced. When replacing the medium, carefully aspirate part of the culture medium and then add an appropriate amount of fresh culture medium. After 7-10 days of culture, if a large number of empty plaques are found in the cells in the culture dish, or the cells are obviously floating (see Figure 6 ), at which point the adenovirus can be collected.

[0064] 2. Collection and amplification of recombinant adenovirus rAd5TsCLP

[0065] Carefully aspirate the cell culture medium obtained from the adenovirus packaging in step 1, avoiding touching the cells, scrape the cells after adding 0.5 mL PBS, and transfer to a 1.5 mL centrifuge tube. Use the repeated freeze-thaw method to lyse the cells, specifically, freeze and thaw four times at -80℃ and 37℃. Centrifuge after freeze-thaw, and take the supernatant as the P0 generation adenovirus, which can be used for subsequent amplification and purification, and store at -80℃ after aliquoting. Infect HEK293A cells with P0 generation adenovirus to amplify the virus, specifically, add the P0 generation virus to fresh cell culture medium for a small amount of amplification, until the cells show plaques, collect and freeze-thaw four times to obtain P1 generation virus. Infect for three consecutive generations to P4 generation for large-scale amplification, collect, purify and concentrate the virus after plaques are formed. After measuring the titer, aliquot the recombinant adenovirus rAd5TsCLP and store it at -80℃.

[0066] 3. Identification of recombinant adenovirus rAd5TsCLP

[0067] (1) Titer determination of recombinant adenovirus

[0068] The concentration was 1×10 5 / mL HEK293A cells were inoculated in a 96-well plate. After culturing for 24 hours, the recombinant adenovirus rAd5TsCLP virus solution was taken out for 10-fold serial dilution, and then added to the 96-well plate containing HEK293A cells. Eight parallel test wells were set for each dilution gradient, and wild-type adenovirus Ad5Neg was inoculated into HEK293A cells in the same way, and a blank control was set up at the same time. After virus infection, the culture was continued for 96 hours, during which the cell lesions and fluorescence expression were observed, and the virus titer at different time points was calculated using the Reed-Muench method. The titer test results are shown in Figure 7 .Depend on Figure 7 It can be seen that 96 hours after infection, there was no significant difference in the viral titer between the recombinant adenovirus rAd5TsCLP and the wild-type adenovirus Ad5Neg, and the growth trends were consistent, indicating that the insertion of exogenous proteins did not affect the growth performance of the recombinant adenovirus.

[0069] (2) PCR identification of recombinant adenovirus genome

[0070] The recombinant adenovirus rAd5TsCLP genome was extracted from the supernatant of infected cells using a virus gene extraction kit according to the kit instructions. The recombinant adenovirus genome was then verified by PCR. The results showed that the size of the amplified product was 1218 bp, which was consistent with the expected size of the target gene (Ts-CLP gene) (see Figure 8 ).

[0071] (3) Western blot analysis of recombinant adenovirus Ts-CLP protein expression

[0072] The supernatant and lysate of the virus packaging cells were collected (10 μL of 100 mM PMSF was added to 1 mL of RIPA lysate to prepare the lysis working solution). The protein samples were separated by SDS-PAGE, and then the methanol-activated PVDF membrane was 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 with anti-Ts-CLP mouse monoclonal antibody (1:2000 dilution) at 4°C overnight. After washing with TBST three times, goat anti-mouse IgG (H+L) HRP antibody was incubated at room temperature for 2 h, washed with TBST five times, and tested using a UVP Chemstudio instrument. The results showed that the size of the Ts-CLP protein was 45.9 kDa, which was consistent with the expected target band size (see Fig. 9 ).

[0073] (4) Transmission electron microscopy morphology observation

[0074] The virus liquid of adenovirus Ad5Neg and recombinant adenovirus rAd5TsCLP was dropped on a 200-mesh electron microscope grid (copper grid), stained with 1% phosphotungstic acid for 1 min, and the excess liquid was removed. The particles of the two adenoviruses were observed using a transmission electron microscope. The results showed that the recombinant adenovirus rAd5TsCLP presented a regular icosahedral morphology, similar to the empty vector Ad5Neg virus particles. This indicates that after the exogenous target gene is inserted, the adenovirus can be packaged normally and maintain its natural morphology (see Fig.10 ).

[0075] Example 3: Use of recombinant adenovirus carrying Trichinella spiralis CLP gene in vaccine preparation

[0076] In order to determine whether the prepared recombinant adenovirus carrying the Trichinella spiralis CLP gene can be used to prepare a vaccine for trichinosis, the immune effect of the recombinant adenovirus in BALB / c mice was evaluated. The specific method is as follows:

[0077] The present invention adopts two methods of intramuscular injection (IM) and intranasal immunization (IN) to inoculate mice with recombinant adenovirus rAd5TsCLP for animal immunization. Then, in order to evaluate the immune effect of the recombinant adenovirus rAd5TsCLP on mice, the number of intestinal adults 3 days after the mice are orally infected with Trichinella spiralis muscle larvae and the number of muscle larvae 5 weeks later (expressed as the number of larvae per gram of muscle LPG) are detected and the worm reduction rate is calculated. At the same time, blood is collected at 0 weeks, 2 weeks and 7 weeks after the first immunization to detect the levels of specific antibodies (IgG, IgG1, IgG2a, IgM, IgA) and neutralizing antibody NAb in the mouse serum, as well as the levels of total sIgA, specific sIgA and histamine in the intestinal lavage fluid.

[0078] 1. Immunization of animals

[0079] 10 were immunized by intramuscular injection and nasal drops respectively. 8 PFU recombinant adenovirus rAd5TsCLP was inoculated into 6-8 week old BALB / c mice (10 mice per group), and the adenovirus Ad5Neg was used as the vaccine control, and the PBS was used as the blank control. To evaluate the immune effect, two weeks after the first immunization, the mice were orally infected with 250 Trichinella spiralis muscle larvae / mouse.

[0080] 2. Detection of the number of larvae in mouse muscles and adults in the intestine

[0081] The mice were euthanized on the 3rd day and 5th week after infection, and the number of intestinal adults and muscle larvae in mice was determined, and the worm reduction rate was calculated. The number of muscle larvae in mice was determined by pepsin digestion method, as follows: pepsin and hydrochloric acid were added to distilled water at 37°C to prepare digestion solution, and the final concentration of pepsin and hydrochloric acid was 1%. Each mouse was digested with 500 mL of digestion solution, placed in a 37°C incubator with a magnetic stirrer, and digested for 2 h. After the entire digestion process was completed, the residue in the digestion solution was filtered out with a 60-mesh sieve, and allowed to stand at room temperature for 1 h. The upper layer of liquid was gently extracted with a sterile syringe, 100 mL of liquid was retained, 400 mL of distilled water was added again, and repeated washing was performed. Finally, the liquid was transferred to a 50 mL beaker, and the muscle larvae of Trichinella spiralis were counted under a microscope.

[0082] The calculation method of muscle larvae reduction rate is as follows:

[0083] According to the number of muscle larvae in each mouse, the number of muscle larvae per gram of muscle of each mouse (LPG) was calculated, and the average LPG of each group of mice was counted and compared with the LPG of the PBS control group, and the worm reduction rate of each experimental group was calculated. The formula is as follows: Muscle larvae reduction rate (%) = (1-average LPG of experimental group / LPG of PBS control group) × 100%.

[0084] Test results such as Fig.11 As shown in Figure 2, 5 weeks after the infection, the muscle larval loads in the IM, IN and Ad5Neg empty vector groups were reduced by 45.61%, 47.91% and 10.57% compared with the PBS group, indicating that different inoculation methods of the recombinant virus rAd5TsCLP all produced a worm reduction rate in mice and played a protective effect (see Figure 2). Fig.11In addition, compared with the PBS group, the adult load of mice immunized with IM, IN and Ad5Neg empty vector groups on the third day after Trichinella infection was reduced by 36.49%, 42.57% and 10.59%, respectively. It can be seen that for the intestinal stage, compared with intramuscular injection, nasal drop immunization provides better protection, and there is no significant difference between the PBS group and the Ad5Neg group (see Fig.11 B in the figure).

[0085] 3. Detection of specific antibody levels in mouse blood

[0086] Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect specific antibodies (IgG, IgG1, IgG2a, IgM, and IgA) in the serum of each group of mice. The specific method was as follows: the purified recombinant protein rTs-CLP at a concentration of 5 μg / mL was coated on a 96-well plate at 4°C overnight; then 100 μL / well blocking solution (5% skim milk powder) was added and blocked at 37°C for 1 h; the collected serum was diluted 10-fold in series and incubated at 37°C for 1 h; goat anti-mouse IgG-HRP was added for incubation; finally, TMB substrate colorimetric solution was added for color development, the reaction was terminated with 2 M H2SO4 sulfuric acid, and the OD was measured using an enzyme reader. 450 value.

[0087] The results showed that 2 weeks after the first immunization, the IgG antibody titers of the IM and IN groups showed a significant increasing trend within 2 weeks compared with the PBS and Ad5Neg groups. The serum to be tested was diluted 50 times to detect the levels of IgG1, IgG2a, IgM and IgA specific antibodies. It was found that there was no significant difference between the PBS group and the Ad5Neg group at each test time point. After 5 weeks of attack (7 weeks of immunization), the anti-Ts-CLP IgG1, IgG2a, IgM, and IgA antibody levels of the two control groups increased. Compared with the control group, the IgG1, IgG2a, IgM, and IgA antibody levels in the IM group were significantly improved. The IN group can also induce antibody levels comparable to those of the IM group, and compared with the IM group, the IN group can induce higher titers of mucosal immunity-related IgA antibodies (see Fig.12 ).

[0088] 4. Neutralizing antibody detection in mouse blood

[0089] The collected sera were 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 DMEM) and mixed with an equal volume of 100 TCID 50The recombinant adenovirus rAd5TsCLP was mixed and incubated at 37°C for 1 h, and then 200 μL of the serum-virus mixture was added to the monolayer of HEK293A cells. HEK293A cells treated with recombinant adenovirus Ad5Neg and not inoculated were used as the control group. The cells were cultured at 37°C and 5% CO2 for 72 hours, the expression of viral fluorescent protein was observed, and the neutralizing antibody titer was calculated. If the number of green fluorescent foci in the sample dilution solution was reduced by more than 50% compared with the infected control group, the neutralizing antibody was judged to be positive. The anti-rAd5TsCLP neutralizing antibody titers in the IM group and the IN group showed a significant increasing trend 2 weeks after immunization (see Fig.13 ).

[0090] 5. Analysis of sIgA and histamine levels in mouse intestine

[0091] After the mice were euthanized, the small intestine was removed and the intestine was repeatedly flushed (intestinal flushing fluid: PBS buffer containing 1% EDTA and PMSF). The intestinal flushing fluid was collected into an EP tube, centrifuged at 4°C for 5 min, and the supernatant was separated. The total sIgA and specific sIgA of Trichinella spiralis were detected by indirect ELISA method. The specific method was as follows: the crude protein of Trichinella spiralis and Ts-CLP protein were coated on the ELISA plate, the primary antibody was the intestinal flushing fluid, the secondary antibody was goat anti-mouse IgA, and the OD value was measured at 450nm. The level of histamine in the intestinal fluid was detected at 0, 2, and 7 weeks after immunization, and the concentration of histamine in the intestine was detected using a mouse enzyme-linked immunosorbent assay kit. The results showed that the levels of total sIgA and Ts-CLP-specific sIgA antibodies in the intestinal lavage fluid could be detected five weeks after the attack (7 weeks of immunization). In addition, the IN group could stimulate higher levels of total sIgA and Ts-CLP-specific sIgA than the IM group. The IN group showed higher histamine levels than the IM group. After 5 weeks of infection (7 weeks of immunization), the histamine level returned to the normal level of the control group. The IN group can cause more obvious intestinal mucosal reactions and histamine secretion levels compared with the IM group (see Fig.14 ).

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant adenovirus vector vaccine for preventing and treating trichinellosis, characterized in that: The recombinant adenovirus vector vaccine is obtained by constructing a recombinant adenovirus vector carrying the Trichinella CLP gene and then transfecting mammalian cells; the recombinant adenovirus vector is obtained by homologous recombination of a replication-deficient adenovirus backbone plasmid and a recombinant adenovirus shuttle plasmid carrying the Trichinella CLP gene; the replication-deficient adenovirus backbone plasmid is pAdEasy-1, and the nucleotide sequence of the Trichinella CLP gene is shown in SEQ ID NO.1; the immunization method of the recombinant adenovirus vector vaccine is intramuscular injection or nasal drops, and the number of vaccinations is 1 dose.

2. The recombinant adenovirus vector vaccine according to claim 1, characterized in that The method for constructing the recombinant adenovirus vector comprises the following steps: (1) using primers with nucleotide sequences such as SEQ ID NO.2 and SEQ ID NO.3 to amplify and obtain a Trichinella spiralis CLP gene fragment; (2) ligating the Trichinella spiralis CLP gene fragment obtained by amplification in step (1) into a shuttle plasmid, and introducing the obtained ligation product into Escherichia coli for amplification, and extracting and obtaining a recombinant shuttle plasmid; (3) The recombinant shuttle plasmid extracted in step (2) is subjected to single enzyme digestion to obtain a linearized recombinant shuttle plasmid, and the linearized recombinant shuttle plasmid is transformed into Escherichia coli BJ5183 carrying the pAdEasy-1 plasmid for homologous recombination, and the recombinant adenovirus vector is obtained by kanamycin screening.

3. The recombinant adenovirus vector vaccine according to claim 2, characterized in that: In step (2), the shuttle plasmid is pShuttle-CMV, and the Escherichia coli is Escherichia coli DH5α.

4. The recombinant adenovirus vector vaccine according to claim 1, characterized in that The mammalian cells are HEK293A cells.

5. The recombinant adenovirus vector vaccine according to claim 1, characterized in that: The recombinant adenovirus vector vaccine is prepared by linearizing the recombinant adenovirus vector and then transfecting it into mammalian cells, packaging and amplifying the recombinant adenovirus, collecting the diseased cells, and repeatedly freezing and thawing to obtain the recombinant adenovirus, namely, the recombinant adenovirus vector vaccine.

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