CN117883561B - Preparation and application of avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine - Google Patents

Abstract

The invention relates to preparation and application of an avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine, wherein the preparation of the nanoparticle vaccine comprises the following steps: (1) preparing Fiber2 protein of avian adenovirus serum type 4; (2) MSN-CC nanoparticle preparation; (3) preparing MSN-CC-Fiber2 nanometer vaccine. The MSN-CC synthesized based on the MSN technology has the characteristics of large aperture, high load and the like, and can not only improve the load capacity of macromolecular proteins, but also improve the utilization rate of antigens. The research synthesizes MSN-CC nano-particles with large aperture and high pore volume, and loads the purified virus protective antigen Fiber2 protein into the MSN-CC nano-particles to prepare the MSN-CC-Fiber2 nano-vaccine. The nano vaccine has good immune protection effect, and MSN-CC nano particles have adjuvant effect.

Description

Preparation and application of avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine

Technical Field

The invention relates to the field of poultry pathology, in particular to preparation and application of an avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine.

Background

Avian adenovirus serotype 4 (Fowl adenovirus serotype-4, FAdV-4), a member of the genus avian adenovirus, is the major epidemic pathogen in farms, causing significant economic losses to the poultry industry. Novel FAdV-4 shows novel genomic features such as natural deletion of open reading frames ORF19 and ORF 27, most importantly, the novel FAdV-4 strain has strong pathogenicity to chicken and mortality rate is as high as 100%. At present, FAdV-4 strains with strong pathogenicity are often found in farms, and the toxicity to chicken industry is extremely great. Thus, there is an urgent need to develop effective vaccines that can control the occurrence of the novel FAdV-4 disease, and although inactivated vaccines have been developed, so far no nanovaccine has been commercialized against the novel strain.

MSN has unique divergent pore canal structure characteristics, biodegradability and good thermal stability, is an ideal non-viral vector for drug delivery, and has good tolerance in a mammalian system, so MSN has high application potential in future vaccine development, which provides possibility for developing FAdV-4 nanoparticle vaccines based on MSN, however, at present, MSN nanoparticles are mainly used as vectors for drugs, and have less research on carrying antigen proteins. Meanwhile, the particle size, the pore diameter and the like of the nano particles prepared by different preparation methods have great difference, and once the pore diameter or intermolecular acting force of the nano particles is not matched with the antigen to be loaded, the antigen can not be loaded or can be loaded but the loading rate is not high, or the nano particles can be loaded but the medicine can not be released at a constant speed, so that the slow release effect can not be achieved. Like conventional MSN nanoparticles (such as the MSN nanoparticle disclosed in patent 201911327322.6) it is capable of supporting DLDH protein, but it is not a universal nanoparticle with very low loading rate for avian adenovirus serotype 4 Fiber2 protein. Based on the above, it is necessary to develop an MSN nanoparticle vaccine capable of effectively loading avian adenovirus serum type 4 Fiber2 protein, and the vaccine has a slow release effect.

Disclosure of Invention

The invention aims to provide an avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine with good immune protection effect and slow release effect, and a preparation method and application thereof.

The aim of the invention is realized by the following technical scheme: a preparation method of an avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine comprises the following steps:

(1) Preparing Fiber2 protein of avian adenovirus serum type 4;

(2) MSN-CC nanoparticle preparation: adding cetyl trimethyl ammonium chloride, triethanolamine and ultrapure water into a three-neck flask, and stirring at 60 ℃ for 1h at 60r/min to completely dissolve the cetyl trimethyl ammonium chloride; then quickly adding an organic mixture, wherein the organic mixture is formed by mixing tetraethyl orthosilicate and chlorobenzene according to the volume ratio of 1:7, then adding ultrapure water, and stirring and mixing at 60 ℃ for reaction for 12 hours at 60 r/min; centrifuging at 8000rpm for 10min after the reaction is finished, and discarding the supernatant; collecting the product, drying at 60 ℃ for 15-20h until the product is completely dried, then transferring the product into a tube furnace, calcining at 550 ℃ for 4h, grinding the product into powder to obtain MSN-CC nano-particles, and drying and storing the MSN-CC nano-particles in a refrigerator at 4 ℃;

wherein chlorobenzene is used to regulate MSN-CC nanoparticle pores.

(3) Preparing MSN-CC-Fiber2 nanometer vaccine: fiber2 protein is loaded by a physical adsorption mode, and the protein concentration is 4.2mg/mL; before use, MSN-CC nanoparticle powder is resuspended into 1mg/mL by using pH8.0 Tris buffer solution, and is uniformly dispersed by ultrasonic treatment for 10min under ice bath condition, then Fiber2 protein is mixed with the resuspended MSN-CC nanoparticle, and the mixture is placed on a rotary mixer to be gently mixed uniformly, so that the MSN-CC nanoparticle and the Fiber2 protein are fully contacted, adsorbed for 12-16h, the Fiber2 protein is loaded into the gaps of the MSN-CC nanoparticle, centrifuged for 10min at 8000rpm, washed twice by using deionized water, the non-adsorbed Fiber2 protein is removed, and the avian adenovirus serum 4-type MSN-CC-Fiber2 nanoparticle vaccine is obtained after vacuum freeze drying.

In the step (2), the volume ratio of the hexadecyl trimethyl ammonium chloride to the triethanolamine to the organic mixture is 2.5-3:0.1-0.2:1.

Preferably, in the step (2), the volume ratio of the cetyltrimethylammonium chloride, the triethanolamine and the organic mixture is 2.73:0.1:1.

In step (2), the pH8.0 Tris buffer, containing 500mM NaCl with 5% glycerol.

The preparation method further comprises a step (4), wherein the step (4) specifically comprises the following steps: and (3) re-suspending the avian adenovirus serum 4 type MSN-CC-Fiber2 nano-particle vaccine precipitate obtained in the step (3) in PBS buffer solution with pH of 7.4, and finally preparing the MSN-CC-Fiber2 nano-vaccine suspension with the concentration of 250 mug/mL.

The pH7.4 PBS buffer contained 150mM NaCl, 5% glycerol.

In the preparation method, the specific method of the step (1) is as follows:

1.1fiber2 Gene construction:

1.1.1 amplification of the Fiber2 gene of avian adenovirus serotype 4 by PCR, wherein the upstream primers used in the amplification were: 5'-ATGAAAAAGACAGCTATCGCGATTGC-3', the downstream primer sequence is: 5'-TCAGTGGTGGTGGTGGTGGTGCTC-3';

1.1.2PCR double enzyme digestion of the product: cutting the PCR product obtained in the step 1.1.1 by HindIII enzyme and XhoI enzyme at 37 ℃ for 60min, performing agarose gel nucleic acid electrophoresis after enzyme cutting, observing whether enzyme cutting is complete or not under a gel imaging system, cutting a target fragment next to an electrophoresis strip, and then performing gel recovery and purification;

1.1.3 vector plasmid pET32a-TEV double cleavage: the vector plasmid pET32a-TEV is subjected to enzyme digestion by using HindIII enzyme and XhoI enzyme;

1.1.4 connecting the vector skeleton after double enzyme digestion in the step 1.1.3 and the gene obtained in the step 1.1.2, wherein a connecting reaction system is 10 xT 4 DNA LIGASE Buffer 1 mu L, T4 DNA LIGASE 1 mu L,1 mu L of vector skeleton and 7 mu L of gene, and connecting at 16 ℃ for overnight to obtain a pET32a-SIP connecting product;

1.1.5 adding the pET32a-SIP connection product obtained in the step 1.1.4 into competent cells BL21 (DE 3), placing on ice, heating in a water bath at 42 ℃ for 45sec, immediately transferring to ice, and cooling for 2min; adding 500 mu L of liquid LB culture medium without any antibiotics, resuscitating for 1h on a shaking table, coating a plate, and culturing for 14h at 37 ℃ on a flat plate; after the colony is picked up by the flat plate for culture, extracting plasmids for double enzyme digestion verification and sequencing verification, and freezing and storing positive recombinant bacteria;

1.2fiber2 protein expression and purification

Thawing the pET-32a-Fiber2 expression bacteria obtained in the step 1.1, sucking 10 mu L to 1mL of liquid LB culture medium, shaking overnight at 37 ℃ and 220rpm, fully resuscitating recombinant bacteria, and then inoculating the recombinant bacteria into 10mL of liquid LB culture medium according to the ratio of 1:1000, and continuously culturing for 6 hours at 37 ℃ and 200 rpm; inoculating into 1L of liquid LB according to a ratio of 1:1000, continuously culturing for 3.5 hours at 37 ℃ and 200rpm, setting the temperature of a shaking table at 16 ℃ for induction overnight, adding IPTG into the bacterial liquid for induction culture overnight after cooling, and shaking for over night at 190 rpm; pouring the bacterial liquid after the induction culture into a bacterial collecting bottle, centrifuging at the temperature of 4 ℃ and at the speed of 8000rpm for 10min, and collecting sediment; re-suspending and precipitating with protein buffer solution, crushing thalli with a high-pressure cell crusher, respectively washing 1 time with ddH ₂ O and 2 times with protein buffer solution for balancing the high-pressure cell crusher before crushing, crushing at 5 ℃ under 700bar for 2min, centrifuging at 12000 Xg for 45min after high-pressure crushing, collecting supernatant, adding the supernatant into a nickel column after balancing with the protein buffer solution, and ensuring that target protein is fully combined with nickel at a flow rate of 6 s/drop; eluting with 20mM imidazole and 40mM imidazole to remove the impurity protein, and eluting with 300mM imidazole to obtain target protein with an elution speed of 10 s/drop; adding TEV enzyme into the collected protein suspension, and performing enzyme digestion and dialysis at 4 ℃ for overnight; and (3) passing the dialyzed protein through a nickel column for the 2 nd time, collecting a flow-through liquid, adding a protein buffer containing 10mM imidazole to elute Fiber2 protein on the column after the flow-through liquid is finished, and concentrating the protein to 4.2mg/mL.

The invention also provides the avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine prepared by the preparation method.

The avian adenovirus serum type 4 MSN-CC-Fiber2 nanoparticle vaccine is applied to the preparation of avian adenovirus serum type 4 vaccine.

Compared with the prior art, the invention has the advantages that:

The MSN-CC synthesized based on the MSN technology has the characteristics of large aperture, high load and the like, and can not only improve the load capacity of macromolecular proteins, but also improve the utilization rate of antigens.

The research synthesizes MSN-CC nano-particles with large pore canal and high load capacity compared with the traditional MSN nano-particles, and loads the purified virus protective antigen Fiber2 protein into the MSN-CC nano-particles to prepare the MSN-CC-Fiber2 nano-vaccine. The toxicity attack protection test result shows that the nano vaccine MSN-CC-Fiber2 has good immune protection effect, and the MSN-CC-Fiber2 nano vaccine added with the MSN-CC carrier has better immune protection effect than pure protein Fiber2, and the MSN-CC nano particle carrier has adjuvant effect, which lays a foundation for developing the avian adenovirus 4 type nano particle vaccine based on MSN-CC in the future. In addition, according to the vaccine release test result, the vaccine prepared by the invention can be released slowly and continuously, and SDS-PAGE proves that Fiber2 protein released from supernatant of vaccine suspension is uniform and stable, and no degradation phenomenon occurs, which indicates that the vaccine prepared by the invention has a slow release effect.

Drawings

FIG. 1 is an agarose nucleic acid electrophoresis diagram in the construction process of a recombinant plasmid, wherein FIG. 1A is an electrophoresis diagram after Fiber2 gene amplification, M, DL2000 bp Marker; lane 1, fiber gene (1509 bp); FIG. 1B is an electrophoretogram identified by double restriction enzyme for recombinant plasmid, lane 1, pET-32a vector backbone (5876 bp) and the target gene band (1509 bp).

FIG. 2 is an electrophoretogram of Ni-NTA purified Fiber2 protein; wherein M: a Marker;1: expressing a bacterial lysate supernatant; 2: flowing through liquid; 3: eluting the hybrid protein by 20mM imidazole; 4: eluting the target protein by 300mM imidazole; 5:300mM eluted protein+TEV enzyme; 6: passing through the nickel column flow at the 2 nd time; 7, loading the solution on a nickel column of 10mM imidazole eluent for the 2 nd time; 8: TEV enzyme.

FIG. 3 is a graph showing the measurement results of the loading rates of MSN-CC and MSN on Fiber2 protein, respectively.

FIG. 4 is a graph showing the results of the MSN-CC-Fiber2 nanovaccine release assay of the present invention.

FIG. 5 is a graph showing the results of an attack protection test for MSN-CC-Fiber2 nanovaccines of the present invention.

Detailed Description

The present invention is described in detail below with reference to the drawings and examples of the specification:

1 Experimental method

1.1Fiber2 Gene construction

The Fiber2 gene was amplified by PCR, the PCR reaction system and procedure are shown in tables 1 and 2, and the upstream primers are: 5'-ATGAAAAAGACAGCTATCGCGATTGC-3' (SEQ ID NO. 1), the downstream primer sequence is: 5'-TCAGTGGTGGTGGTGGTGGTGCTC-3' (SEQ ID NO. 2). PCR product double cleavage System: the total volume was 50. Mu.L, of which 10X Cutsmart buffer. Mu.L, hindIII 1. Mu.L, xhoI 1. Mu.L, 20. Mu.L of the purified PCR product and ddH ₂ O23. Mu.L. And (3) performing enzyme digestion at 37 ℃ for 60min, performing agarose gel nucleic acid electrophoresis (agarose concentration is 1.2%) after enzyme digestion, observing whether enzyme digestion is complete or not under a gel imaging system, cutting a target fragment next to an electrophoresis strip, and then performing gel recovery and purification. The cleavage system for the vector plasmid pET32a-TEV is as follows: the total volume was 50. Mu.L, of which 10X Cutsmart buffer. Mu.L, hindIII 1. Mu.L, xhoI 1. Mu.L, vector plasmid 5. Mu.L, ddH ₂ O38. Mu.L, and the gel was recovered to give a vector backbone. The double digested vector backbone was ligated to the gene in a 10 XT 4 DNA LIGASE Buffer 1. Mu.L, T4 DNA LIGASE. Mu.L, 1. Mu.L vector backbone, 7. Mu.L gene, and overnight at 16 ℃. Adding the obtained pET32a-SIP connection product into competent cells BL21 (DE 3), placing on ice, heating in a water bath at 42 ℃ for 45sec, immediately transferring to ice, and cooling for 2min; after resuscitating on a shaker for 1h, 500. Mu.L of liquid LB medium without any antibiotics was added, and the plates were incubated at 37℃for 14h. After the colony is picked and cultured by the flat plate, plasmids are extracted for double enzyme digestion verification and sequencing verification, and positive recombinant bacteria are frozen.

TABLE 1PCR reaction System

Table 2 PCR reaction procedure

98℃	30sec	×1
			98℃	12sec
60℃	25sec	×35cycles
			72℃	6min
72℃	10min	×1
			10℃	∞

1.2 Fiber2 protein expression and purification

After thawing the pET-32a-Fiber 2-expressing strain, 10. Mu.L to 1mL of liquid LB medium (containing 100. Mu.g/mL of ampicillin) was aspirated, and the strain was shaken overnight at 37℃and 220rpm to allow the recombinant strain to recover sufficiently, followed by inoculating the strain into 10mL of liquid LB medium (containing 100. Mu.g/mL of ampicillin) at 1:1000, and culturing continuously at 200rpm for 6 hours at 37 ℃. Inoculating into 1L of liquid LB according to a ratio of 1:1000, continuously culturing at 37 ℃ and 200rpm for 3.5 hours, setting the temperature of a shaking table at 16 ℃ for induction overnight (12-16 hours), adding IPTG (final concentration is 0.3 mM) into the bacterial liquid after cooling for induction culture overnight, and shaking at 190rpm for overnight. Pouring the bacterial liquid after the induction culture into a bacterial collecting bottle, centrifuging at the temperature of 4 ℃ and at the speed of 8000rpm for 10min, and collecting the precipitate. The pellet was resuspended in protein buffer (25 mM Tris pH 8.0, 500mM NaCl, 0.5% Tween, 2mM beta-mercaptoethanol, 5mM imidazole), the cells were broken with a high pressure cell breaker, the cells were equilibrated with ddH ₂ O1 pass before disruption, the protein buffer 2 passes before disruption, the disruption conditions were 5 ℃, the cells were broken at 700bar for 2min, after the end of high pressure disruption, the supernatant was collected by 12000 Xg centrifugation for 45min, and the supernatant was added to a nickel column after equilibration of the protein buffer at a flow rate of 6 s/drop to allow the target protein to bind well to nickel. Then eluting with 20mM imidazole and 40mM imidazole to remove the impurity protein, and then eluting with 300mM imidazole to obtain the target protein with the elution speed of 10 s/drop. The collected protein suspension was then added to TEV enzyme and dialyzed overnight at 4 ℃. And (3) passing the dialyzed protein through a nickel column for the 2 nd time, collecting a flow-through liquid, adding a protein buffer containing 10mM imidazole to elute Fiber2 protein on the column after the flow-through liquid is finished, and concentrating the protein to 4.2mg/mL.

1.3 MSN-CC nanoparticle preparation

Cetyltrimethylammonium chloride (Hexadecyltrimethylammonium chloride, CTAC) is used as a template agent, tetraethyl orthosilicate (TETRAETHYL ORTHOSILICATE, TEOS) is used as a silicon source, triethanolamine (Triethanolamine, TEOA) is used as a catalyst, and the MSN-CC particles are synthesized by the hydrolysis-condensation process of TEOS under alkaline conditions. The specific process is as follows: CATC 4.8.8 g (10.9 mL), TEOA0.4mL, and ultra-pure water 14.4mL were added together into a 100mL three-necked flask, and stirred at 60℃for 1h at 60r/min to completely dissolve the CTAC template; then 4mL of an organic mixture containing 0.5mL TEOS,3.5mL chlorobenzene (used to modulate MSN-CC nanoparticle porosity) was added rapidly; adding 10mL of ultrapure water, stirring and mixing at 60 ℃ and 60r/min for reaction for 12 hours; centrifuging at 8000rpm for 10min after the reaction is finished, and discarding the supernatant; collecting the product, drying at 60deg.C for 15-20 hr to completely dry, calcining at 550deg.C in a tube furnace for 4 hr to remove template, grinding into powder to obtain MSN-CC nanoparticles, drying, and storing in a refrigerator at 4deg.C.

1.4 Preparation of MSN-CC-Fiber2 nanometer vaccine and measurement of load rate thereof

Fiber2 protein is loaded in a physical adsorption mode, the protein concentration is 4.2mg/mL, before use, MSN-CC nanoparticle powder is resuspended to 1mg/mL by using a pH8.0 Tris buffer (500 mM NaCl, 5% glycerol), and is uniformly dispersed by ultrasonic treatment for 10min under ice bath conditions, then Fiber2 protein and the resuspended MSN-CC nanoparticle powder are mixed at 4 ℃, and the mixture is placed on a rotary mixer to be gently mixed, so that MSN-CC nanoparticle and Fiber2 protein are fully contacted and adsorbed for 12-16h, and Fiber2 protein is loaded into MSN-CC nanoparticle pores. Centrifuging at 8000rpm for 10min, washing twice with deionized water, removing non-adsorbed Fiber2 protein, and vacuum freeze drying to obtain fowl adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine.

Meanwhile, referring to patent 201911327322.6, conventional MSN nanoparticles are prepared and MSN nanoparticles are used as a control of Fiber2 protein carriers (referring to a preparation method of MSN-CC-Fiber2 nanoparticle vaccine, the prepared MSN nanoparticles are mixed with Fiber2 to prepare MSN-Fiber2 nanoparticle vaccine), and the difference of MSN-CC nanoparticles and MSN loading rates is compared. Then, the MSN-Fiber2 nanometer vaccine and MSN-CC-Fiber2 nanometer vaccine suspension are centrifuged for 10min at 8000rpm under the condition of 4 ℃, the supernatant is collected to measure the concentration of Fiber2 protein and calculate the load rate, and the calculation formula of the Fiber2 load rate is as follows:

Load _Fiber2 = total amount of Fiber2 protein loaded-amount of Fiber2 protein in supernatant

Load ratio = load _Fiber2/(load _Fiber2 +msn-CC mass) ×100%

Finally, the nano vaccine precipitate is resuspended in PBS buffer (150 mM NaCl, 5% glycerol) at pH7.4, and finally the MSN-CC-Fiber2 nano vaccine suspension with the concentration of 250 mug/mL is prepared.

1.5 Vaccine Release test

2ML of MSN-CC-Fiber2 nanoparticle vaccine resuspended in PBS was gently shaken at 37℃and 100r/min for 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, and 0.2mL of suspension was removed for protein concentration determination at each time period. And the suspension was removed and centrifuged at 12000rpm for 20min at 4℃to obtain a supernatant. The release and degradation of Fiber2 protein from MSN-CC at different time intervals was determined by SDS-PAGE.

1.6 Toxicity attack protection test

80 SPF chickens were randomly divided into 4 groups of 20, which are MSN-CC-Fiber2 vaccine group, fiber2 protein group, virus attack group and PBS blank group. Vaccine immunization was 2 times, 7 days old for immunization 1 and 21 days old for immunization 2. Immunization was performed by cervical subcutaneous injection, 200. Mu.L/mouse, 50. Mu.g of vaccine suspension was injected into each of the MSN-CC-Fiber2 vaccine groups, 50. Mu.g of Fiber2 protein was injected into each of the Fiber2 protein groups, and the other two groups were injected with PBS buffer as controls. SPF chicken is infected by leg intramuscular injection FAdV-4 virus suspension at 35 days after vaccine immunization, FAdV-4 virus suspension is injected into MSN-CC-Fiber2 vaccine group, fiber2 group and virus attack group, the virus attack dose is 0.5 mL/dose, the virus attack dose is 0.5X10 ^3.8TCID₅₀/mL, and PBS is injected into control group for 0.5mL. Continuously observing the feed intake and growth condition of each group of SPF chickens for 14 days after the virus attack, recording the survival and death condition of each group of SPF chickens, counting the death rate and the survival rate, and preparing a survival curve. And calculating a relative immune protection Rate (RPS) according to a RPS calculation formula:

Rps= [1- (vaccine immune group mortality/challenge group mortality) ]x100%.

2 Experimental results

2.1Fiber2 Gene PCR amplification and recombinant plasmid construction

The result of PCR amplification showed that the molecular weight of the target gene was 1509bp (FIG. 1A in FIG. 1). The result of double enzyme digestion verification electrophoresis is shown in FIG. 1B of FIG. 1, and agarose nucleic acid electrophoresis after double enzyme digestion is carried out to obtain two bands, namely 1509bp target gene Fiber2 and 5876bp carrier frameworks respectively. The positive clones were sent to the company for detection, and the sequencing result further confirmed that the recombinant plasmid was positive.

2.2 Expression and purification of recombinant proteins

SDS-PAGE identifies the Ni-NTA purified Fiber2 protein, and the Fiber2 recombinant protein is mainly present in the supernatant after disruption of the expressing strain. The 20mM imidazole was eluted to remove the hybrid protein, and then the target protein was eluted with 300mM imidazole to give a protein with Trx+His tag having a molecular weight of about 78kDa. After the TEV was dialyzed overnight by digestion, the Trx+His tag protein was removed and finally the 60kDa protein was collected (FIG. 2 lanes 6 and 7).

2.3MSN-CC-Fiber2 nanovaccine load Rate

The MSN-Fiber2 nano-vaccine and MSN-CC-Fiber2 nano-vaccine precipitate obtained by loading overnight are resuspended in PBS buffer (150 mM NaCl, 5% glycerol) with the final concentration of 250 mug/mL nano-vaccine suspension. The loading rate of MSN-CC nanoparticles was 48.8% by calculation, while the loading rate of MSN nanoparticles was only 6.5% (FIG. 3). The MSN-CC nanoparticles have better loading effect than MSN nanoparticles, and the analysis is mainly because MSN-CC channels are larger than MSN, and are more suitable for loading Fiber2 protein with molecular weight of 60 kDa.

2.4 Vaccine Release test

The release and degradation conditions of the Fiber2 protein from the MSN-CC are measured by SDS-PAGE, the slow and continuous release effects of the Fiber2 protein at 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h are known from the figure, and the SDS-PAGE proves that the Fiber2 protein released from the supernatant of the vaccine suspension is uniform and stable, and the degradation phenomenon does not occur (figure 4).

2.5 Toxicity attack protection test

In order to detect the protective effect of MSN-CC-Fiber2 nanometer vaccine, 35-day-old SPF was detoxified after vaccine immunization. FAdV-4 SPF-chickens were continuously observed for 14 days after infection, and death of each group of SPF-chickens was recorded daily. The death rate of the SPF chickens in the virus attack group is 35% from day 2 to day 7, the death rate is 90% from day 3, and the death rate of the SPF chickens in the Fiber2 protein group is 35% from day 8, but the SPF chickens in the blank control group and the MSN-CC-Fiber2 nano vaccine group do not die. The relative immunoprotection rate of the MSN-CC-Fiber2 nanovaccine group was 100%, while the relative immunoprotection rate of the Fiber2 proteome without MSN-CC nanoparticles was only 61% (FIG. 5). The result shows that the MSN-CC-Fiber2 nano vaccine has good immune protection effect and MSN-CC has good adjuvant effect.

Finally, it should be noted that the embodiments described above are part of the embodiments of the invention and should not be limited in this alignment. Equivalent changes and modifications of the invention are intended to be covered by the present invention, as both the claims and the specification should be interpreted as falling within the scope of the invention.

Claims (9)

1. A preparation method of an avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine is characterized by comprising the following steps: it comprises the following steps:

(1) Preparing Fiber2 protein of avian adenovirus serum type 4;

(2) MSN-CC nanoparticle preparation: adding cetyl trimethyl ammonium chloride, triethanolamine and ultrapure water into a three-neck flask, and stirring at 60 ℃ for 1h at 60r/min to completely dissolve the cetyl trimethyl ammonium chloride; then quickly adding an organic mixture, wherein the organic mixture is formed by mixing tetraethyl orthosilicate and chlorobenzene according to the volume ratio of 1:7, then adding ultrapure water, and stirring and mixing at 60 ℃ for reaction for 12 hours at 60 r/min; centrifuging at 8000rpm for 10min after the reaction is finished, and discarding the supernatant; collecting the product, drying at 60 ℃ for 15-20h until the product is completely dried, then transferring the product into a tube furnace, calcining at 550 ℃ for 4h, grinding the product into powder to obtain MSN-CC nano-particles, and drying and storing the MSN-CC nano-particles in a refrigerator at 4 ℃;

(3) Preparing MSN-CC-Fiber2 nanometer vaccine: fiber2 protein is loaded by a physical adsorption mode, and the protein concentration is 4.2mg/mL; before use, firstly re-suspending MSN-CC nanoparticle powder into 1mg/mL with pH8.0 Tris buffer, carrying out ultrasonic treatment under ice bath condition for 10min to enable the MSN-CC nanoparticle powder to be uniformly dispersed, then mixing Fiber2 protein with the re-suspended MSN-CC nanoparticle powder at 4 ℃, placing the mixture on a rotary mixer for shaking to enable MSN-CC nanoparticle to be fully contacted with Fiber2 protein, adsorbing for 12-16h, enabling Fiber2 protein to be loaded into MSN-CC nanoparticle gaps, centrifuging for 10min at 8000rpm, washing twice with deionized water to remove non-adsorbed Fiber2 protein, and carrying out vacuum freeze drying to obtain the avian adenovirus serum 4 type MSN-CC-Fiber2 nanoparticle vaccine.

2. The method of manufacturing according to claim 1, characterized in that: in the step (2), the volume ratio of the hexadecyl trimethyl ammonium chloride to the triethanolamine to the organic mixture is 2.5-3:0.1-0.2:1.

3. The preparation method according to claim 2, characterized in that: in the step (2), the volume ratio of the cetyltrimethylammonium chloride to the triethanolamine to the organic mixture is 2.73:0.1:1.

4. The method of manufacturing according to claim 1, characterized in that: in step (3), the pH8.0Tris buffer, containing 500mM NaCl with 5% glycerol.

5. The method of manufacturing according to claim 1, characterized in that: the method also comprises a step (4), wherein the step (4) is specifically as follows: and (3) re-suspending the avian adenovirus serum 4 type MSN-CC-Fiber2 nano-particle vaccine precipitate obtained in the step (3) in PBS buffer solution with pH of 7.4, and finally preparing the MSN-CC-Fiber2 nano-vaccine suspension with the concentration of 250 mug/mL.

6. The method of manufacturing according to claim 5, wherein: the pH7.4 PBS buffer contained 150mM NaCl, 5% glycerol.

7. The method of manufacturing according to claim 1, characterized in that: the specific method of the step (1) comprises the following steps:

1.1fiber2 Gene construction:

1.1.1 amplification of the Fiber2 gene of avian adenovirus serotype 4 by PCR, wherein the upstream primers used in the amplification were: 5'-ATGAAAAAGACAGCTATCGCGATTGC-3', the downstream primer sequence is: 5'-TCAGTGGTGGTGGTGGTGGTGCTC-3';

1.1.2PCR double enzyme digestion of the product: cutting the PCR product obtained in the step 1.1.1 by HindIII enzyme and XhoI enzyme at 37 ℃ for 60min, performing agarose gel nucleic acid electrophoresis after enzyme cutting, observing whether enzyme cutting is complete or not under a gel imaging system, cutting a target fragment next to an electrophoresis strip, and then performing gel recovery and purification;

1.1.3 vector plasmid pET32a-TEV double cleavage: the vector plasmid pET32a-TEV is subjected to enzyme digestion by using HindIII enzyme and XhoI enzyme;

1.1.4 connecting the vector skeleton after double enzyme digestion in the step 1.1.3 and the gene obtained in the step 1.1.2, wherein a connecting reaction system is 10 xT 4 DNA LIGASE Buffer 1 mu L, T4 DNA LIGASE 1 mu L,1 mu L of vector skeleton and 7 mu L of gene, and connecting at 16 ℃ for overnight to obtain a pET32a-SIP connecting product;

1.1.5 adding the pET32a-SIP connection product obtained in the step 1.1.4 into competent cells BL21 (DE 3), placing on ice, heating in a water bath at 42 ℃ for 45sec, immediately transferring to ice, and cooling for 2min; adding 500 mu L of liquid LB culture medium without any antibiotics, resuscitating for 1h on a shaking table, coating a plate, and culturing for 14h at 37 ℃ on a flat plate; after the colony is picked up by the flat plate for culture, extracting plasmids for double enzyme digestion verification and sequencing verification, and freezing and storing positive recombinant bacteria;

1.2fiber2 protein expression and purification

Thawing the pET-32a-Fiber2 expression bacteria obtained in the step 1.1, sucking 10 mu L to 1mL of liquid LB culture medium, shaking overnight at 37 ℃ and 220rpm, fully resuscitating recombinant bacteria, and then inoculating the recombinant bacteria into 10mL of liquid LB culture medium according to the ratio of 1:1000, and continuously culturing for 6 hours at 37 ℃ and 200 rpm; inoculating into 1L of liquid LB according to a ratio of 1:1000, continuously culturing for 3.5 hours at 37 ℃ and 200rpm, setting the temperature of a shaking table at 16 ℃ for induction overnight, adding IPTG into the bacterial liquid for induction culture overnight after cooling, and shaking for over night at 190 rpm; pouring the bacterial liquid after the induction culture into a bacterial collecting bottle, centrifuging at the temperature of 4 ℃ and at the speed of 8000rpm for 10min, and collecting sediment; re-suspending and precipitating with protein buffer solution, crushing thalli with a high-pressure cell crusher, respectively washing 1 time with ddH ₂ O and 2 times with protein buffer solution for balancing the high-pressure cell crusher before crushing, crushing at 5 ℃ under 700bar for 2min, centrifuging at 12000 Xg for 45min after high-pressure crushing, collecting supernatant, adding the supernatant into a nickel column after balancing with the protein buffer solution, and ensuring that target protein is fully combined with nickel at a flow rate of 6 s/drop; eluting with 20mM imidazole and 40mM imidazole to remove the impurity protein, and eluting with 300mM imidazole to obtain target protein with an elution speed of 10 s/drop; adding TEV enzyme into the collected protein suspension, and performing enzyme digestion and dialysis at 4 ℃ for overnight; and (3) passing the dialyzed protein through a nickel column for the 2 nd time, collecting a flow-through liquid, adding a protein buffer containing 10mM imidazole to elute Fiber2 protein on the column after the flow-through liquid is finished, and concentrating the protein to 4.2mg/mL.

8. An avian adenovirus serotype 4 MSN-CC-Fiber2 nanoparticle vaccine prepared by the method of any one of claims 1-7.

9. Use of an avian adenovirus serotype 4 MSN-CC-Fiber2 nanoparticle vaccine according to claim 8 in the preparation of an avian adenovirus serotype 4 vaccine.