CN112342149A - Avian influenza virus-like particle assembly expression system and application thereof - Google Patents

Abstract

The invention specifically discloses an avian influenza virus-like particle assembly expression system and application thereof, and the system is designed on the basis of the transformation of a pichia pastoris pPIC9K vector, constructs avian influenza virus HA, M and NA gene expression plasmids, electrically converts three expression plasmids into pichia pastoris in proportion, screens positive recombinants, induces expression and assembles VLPs, extracts and purifies an obtained product, immunizes animals, and can obtain an anti-avian influenza virus specific neutralizing antibody. The invention mainly utilizes a pichia pastoris multicopy integrated vector to express and assemble the avian influenza virus-like particles and utilizes the expression system to carry out the industrial application of the avian influenza vaccine.

Description

Avian influenza virus-like particle assembly expression system and application thereof

Technical Field

The invention relates to the technical field of molecular biology, and particularly discloses an avian influenza virus-like particle expression assembly system and application thereof.

Background

Avian Influenza is an acute, thermal and highly contact infectious disease of chickens, ducks, geese, turkeys, pigeons, wild poultry and the like caused by Influenza A Virus (inflenza A Virus), also called true fowl plague or European fowl plague, which is listed as a legal reported epidemic disease by the International Bureau of zooepidemics and is also listed as an animal infectious disease in China. Avian influenza was first isolated in 1959 from a flock outbreaked in Scotland (A/CK/Scotland/59). Since 1996, our country has seen many outbreaks of avian influenza, and even cross-species transmission to infect humans and other animals. According to the classification of pathogen types, avian influenza can be classified into three types, high, low and non-pathogenic. The highly pathogenic avian influenza subtype H5N1 is well known for high morbidity and mortality, resulting in huge economic and social losses.

The influenza A virus belongs to the genus A of the family Orthomyxoviridae, and the virus particle is polymorphic, wherein the spherical diameter is 80-120 nm, and the virus particle has a capsule membrane and is a single-strand negative strand RNA virus. The genome is divided into 8 segments and encodes 11 proteins. Segment 1, 2, 3 encode polymerase proteins (PB2, PBl and PA), segment 4 encodes Hemagglutinin (HA), segment 5 encodes Nucleoprotein (NP), segment 6 Neuraminidase (NA), segment 7M gene encodes matrix protein M1 and ion channel protein M2, segment 8 NS gene encodes nonstructural protein NSl and NS2 and PBl-F2 proteins involved in inducing apoptosis. Influenza a viruses are classified into 16 HA subtypes and 9 NA subtypes according to their HA and NA antigenicity.

Influenza virus particles can be divided into envelope, matrix protein and core three parts from outside to inside. The core of the virus contains the genomic RNA and its replicase. Genomic single negative strand RNA (ss-RNA) binds to Nucleoprotein (NP) and winds around ribonucleoprotein bodies (RNPs), as well as RNA polymerase responsible for RNA transcription. The matrix protein (M1) constitutes the outer shell backbone of the virus, which also contains a small amount of membrane protein (M2). The matrix protein is tightly bound with the envelope of the virus outermost layer to protect the virus core and maintain the virus space structure. The M2 protein has ions (mainly Na)⁺) The channels and the function of adjusting the pH value in the membrane. The envelope is a phospholipid bilayer membrane wrapped outside matrix protein and is derived from the cell membrane of a host. Besides phospholipid molecules, the envelope also contains two glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), which protrude from the surface of the virus and have a length of about 10-40 nm, and are called spikes. Typically, 400 hemagglutinin spikes and 100 neuraminidase spikes are distributed on the surface of the influenza virus. According to the antigenic change of hemagglutinin and neuraminidase, the strain subtypes are distinguished. HA plays an important role in the process of virus introduction into host cells and is immunogenic, and anti-hemagglutinin antibodies can neutralize influenza virus. NA hydrolyzes the sialic acid, cuts off the final connection between the virus and the host cell, so that the virus can be released from the host cell smoothly to infect other cells. Thus, HA and NA are the two most important antigenic proteins of influenza virus. The M gene comprises two reading frames which respectively code matrix protein M1 and ion channel protein M2, and the full-length M gene is inserted into a multi-copy integration vector transformed yeast for expression, thereby being beneficial to the stable transcription of the M gene, prolonging the half-life period of mRNA and further improving the expression quantity of the M1 protein.

Vaccination is the most effective method for influenza prevention, and the most commonly used vaccine is whole virus inactivated vaccine. In recent years, research shows that influenza virus-like particles (VLPs) have the immune efficacy close to that of inactivated whole viruses, are safer relative to the VLPs, do not need to use live viruses in the production process, have no biological safety problem, and are expected to replace inactivated whole virus vaccines. However, most influenza virus VLPs expression assembly systems adopt mammalian and baculovirus-mediated sf9 cell systems, which results in difficult control of production process and high culture cost.

Pichia pastoris (Pichia pastoris) successfully expresses a large amount of foreign proteins, but the expression system based on the vector pPIC9K mostly integrates single-copy or low-copy number genes, and the expression amount of partial proteins is low. Researchers try to improve the expression level of foreign proteins through metabolic pathway modification. However, there are not many cases of success with viral proteins. In view of this, we carried out multi-copy transformation on pPIC9K vector, inserted 18S rDNA partial sequence as multi-copy recombination homology arm, and the test results supported that the integrated copy number of exogenous gene could be increased, effectively improved the expression quantity of protein, and reduced the production cost.

The experiment constructs HA, M and NA gene expression plasmids of avian influenza virus on the basis of the transformation of a pichia pastoris pPIC9K vector, mixes and electrically transforms the three expression plasmids into pichia pastoris according to a proportion, screens positive recombinant strains, induces, expresses and assembles influenza VLPs, and immunizes animals after extracting and purifying products to obtain the avian influenza virus resistant specific neutralizing antibody.

Disclosure of Invention

One of the purposes of the invention is to overcome the defects of low copy number of the recombinant exogenous gene of the pichia pastoris and low expression level of exogenous protein, provide a pichia pastoris genome insertion region with high copy number and stable integration of the exogenous gene and construct a pichia pastoris vector with a multi-copy integration exogenous gene expression frame.

The second purpose of the invention is to overcome the defects of low expression level and low immunogenicity of HA, M and NA genes of the avian influenza virus in a eukaryotic system, and provide a technical method for expressing HA, M and NA proteins in a yeast body in a high copy integration manner and further assembling the proteins into virus-like particles.

The purpose of the invention is realized by the following technical scheme: a recombinant expression avian influenza H5N1 virus-like particle Pichia pastoris GS115/rAI-a/1(pichia pastoris strain GS115/rAI-a/1), the preservation number of the recombinant strain is CCTCC No: m2020630.

Bacterial preservation description: and (3) classification and naming: pichia pastoris GS115/rAI-a/1 for recombinant expression of avian influenza H5N1 virus-like particles; latin learning name: pichia pastoris strain GS 115/rAI-a/1; the preservation number is: CCTCC No: m2020630; the preservation organization: china center for type culture Collection; address: wuhan university in Wuhan, China, zip code: 430072, telephone: (027)68754052, fax: (027)68754833, E-mail: cctcc @ whu.edu.cn; the preservation date is as follows: 26/10/2020.

A target sequence of a pichia pastoris exogenous gene multicopy integration region is located in the middle of an 18S rDNA gene and is shown in SEQ ID NO. 1.

Designing a primer pair according to the target sequence, and amplifying by a PCR method to obtain the sequence for constructing a Pichia pastoris multicopy integration vector p8.5K; in the primer pair, the upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO. 3.

Further designed, the Pichia pastoris multicopy integration vector p8.5K is obtained by inserting SEQ ID NO.1 between Xba I and Bsp 1047I restriction enzyme sites of the eukaryotic expression vector pPIC 9K.

An avian influenza virus-like particle expression plasmid p8.5K-HA, p8.5K-M and p8.5K-NA constructed by a multi-copy integration vector p8.5K, wherein nucleic acid sequences of hemagglutinin HA genes and M genes, namely including matrix protein M1, ion channel protein M2 and neuraminidase NA genes, are amplified from an avian influenza virus H5N1 genome by an RT-PCR method and respectively correspond to gene sequences shown as SEQ ID No.4, No.5 and No. 6.

Further designed, the expression plasmid p8.5K-HA is obtained by digesting with EcoRV and Not I restriction enzymes respectively and inserting between SnaBI and Not I restriction enzyme cutting sites of the multi-copy integration vector p8.5K.

Further designing, the expression plasmids p8.5K-M and p8.5K-NA are obtained by digesting with SnaBI and Not I restriction enzymes and inserting into the positions between SnaBI and Not I restriction enzyme sites of the multi-copy integration vector p8.5K respectively.

Further designed, the three pairs of primers amplified by the HA, M and NA genes are respectively: an upstream primer SEQ ID NO.7 and a downstream primer SEQ ID NO. 8; secondly, an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO. 10; third, an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO. 12.

According to the technical scheme, the implementation of the invention can achieve the following beneficial effects:

compared with a Pichia pastoris vector pPIC9K, the vector p8.5K allows multiple copies of the foreign gene to be inserted into the yeast gene, and can effectively improve the expression amount of the foreign protein. In the embodiment 1 of the present study, generally, pPIC9K can mediate integration of 1-3 copies of foreign genes, and p8.5k is 10-15 (unpublished test data), so that the expression levels of avian influenza viruses HA, M, and NA are significantly increased, and the virus-like particles can be assembled to greatly increase immunogenicity, significantly reduce production cost, and promote the industrialization of genetic engineering vaccines.

Avian influenza is an acute, febrile and highly contagious disease of poultry, is well known for high morbidity and mortality, and poses serious threat to poultry industry. At present, the disease is mainly prevented by adopting whole virus inactivated vaccine produced from poultry eggs. However, the production cost is high due to the risk of virus escape, serious side effects, the need of three-level protection conditions for biosafety and the like in the production process. Therefore, in the research embodiment 2, pichia pastoris is adopted to express HA, M1 and NA proteins of the avian influenza viruses in a recombinant manner, which is helpful for overcoming the defects, so that the risk of virus escape is avoided, the biological safety three-level protection condition is not required, no poultry egg is used, no redundant protein is generated, the side reaction is light, and the comprehensive production cost is low.

Previous studies have shown that avian influenza virus-like particles can be successfully expressed both in mammalian cells and in cells expressing avian influenza virus-like particles such as Spodoptera frugiperda sf 9. However, the expression vector has long construction process, low stability of the expression process and high cost. Wherein the longer construction process even lags behind the variation speed of the influenza virus, which results in the reduction of the protection rate of the vaccine to the variant strain. In view of the above, in the implementation example 3 of the present study, in the process of extracting and purifying HA, M1 and NA proteins, the purification and assembly are optimized, the assembly rate of virus-like particles is increased, and the cost is further reduced.

Drawings

FIG. 1 shows the restriction enzyme digestion identification of expression plasmid. In the figure, 1, Gene Ruler 1Kb plus DNA Ladder, 2, plasmid p8.5K-NA, 3, plasmid p8.5K-M, 4, plasmid p8.5K-HA, 5, plasmid p8.5K, 6 and plasmid pPIC 9K. FIG. 2 shows PCR identification of positive recombinant strains, i.e., PCR identification of HA, M and NA genes of recombinant strains. FIG. 1, Gene Ruler 1Kb plus DNA Ladder; 2. HA gene in p8.5k recombinant GS115 genome; 3. the M gene in the p8.5k recombinant GS115 genome; 4. NA gene in p8.5k recombinant GS115 genome; 5. HA gene in recombination strain rAI-a/1 genome; 6. m gene in recombinant strain rAI-a/1 genome; 7. NA gene in recombination strain rAI-a/1 genome; 8. HA gene in recombinant strain rAI-a/2 genome; 9. m gene in genome of recombination strain rAI-a/2; 10. the NA gene in the genome of the recombinant strain rAI-a/2. FIG. 3 is an SDS-PAGE electrophoretogram of the recombinant protein. In the figure, 1, GS115 yeast protein; 2. p8.5k recombinant GS115 yeast protein; 3. an avian influenza virus antigen; 4. recombinant strain rAI-a/2 sucrose density gradient centrifugation protein; 5. the recombinant strain rAI-a/2 ammonium sulfate precipitates protein; 6. the recombinant strain rAI-a/1 ammonium sulfate precipitates protein; 7. and (3) a protein Marker. FIG. 4 is a recombinant protein immunoblot. In the figure, 1, GS115 yeast protein; 2. p8.5k recombinant GS115 yeast protein; 3. an avian influenza virus antigen; 4. recombinant strain rAI-a/2 sucrose density gradient centrifugation protein; 5. the recombinant strain rAI-a/2 ammonium sulfate precipitates protein; 6. the recombinant strain rAI-a/1 ammonium sulfate precipitates protein; 7. and (3) a protein Marker. FIG. 5 shows the virus-like particles observed by electron microscopy.

Detailed Description

All reagents, materials and equipment used in the present invention are well known in the art and are not intended to limit the practice of the present invention, and other reagents and equipment well known in the art may be used in the practice of the following embodiments of the present invention.

The first embodiment is as follows: pichia pastoris multi-copy integration vector and expression plasmid construction

1. Multi-copy integration vector construction

Designing a primer, as shown in SEQ ID NO.3 and SEQ ID NO.4, taking a Pichia pastoris GS115 strain 18S rDNA sequence (GenBank: FN392325.1) as a template, carrying out PCR amplification on a 1292bp partial sequence, recovering a product from a gel, connecting the product to a pMD19-T vector, and carrying out enzyme digestion on a Kpn21 to identify a plasmid; sequencing of positive plasmid by Competition Biotechnology engineering (Shanghai) Ltd. Xba I and Bsp 1407I share Tango buffer to digest positive pMD19-T-1292bp plasmid and vector pPIC9K, electrophoresis gel is recovered for 1292bp and 7.01Kb respectively, T4 DNA ligase is connected to transform DH5 alpha competent cells, positive colonies are screened on an ampicillin LB plate, plasmids are extracted, Not I enzyme digestion identification is carried out, sequencing is carried out on the positive plasmids by committee biotechnology (Shanghai) Limited, and the positive plasmids are named as multi-copy integration vector p8.5K. The PCR reaction system comprises: 10 XEx Taq Buffer5 μ L; 2.5mmol/L dNTP (10mmol/L) 2.5. mu.L; 1.0 μ L of DNA template; 2.5. mu.L of upstream and downstream primers (10 pmol/uL); ex Taq DNA polymerase 1.0. mu.L; deionized water 35.5. mu.L. The PCR amplification reaction conditions are as follows: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 50 ℃ for 15 seconds, 72 ℃ for 90 seconds; extension at 72 ℃ for 10 min.

2. Acquisition of HA, M and NA genes of avian influenza virus

The A/Pigel/Xinjiang/YL/2018 virus genome RNA is extracted by a Trizol method, and the virus genome RNA is kept at the temperature of-20 ℃ for standby. Three pairs of primer pairs are designed according to the genome sequence of the avian influenza virus reference strain A/LGD/1/9(GenBank: JX 486550): (1) no.7 and No. 8; (2) no.9 and No. 10; (3) no.11 and No.12, as shown in Table 1. Respectively amplifying HA, M and NA genes of structural proteins, and synthesizing by the corporation of Venezetian Biotechnology engineering (Shanghai). The one-step RT-PCR reaction conditions were as follows: at 55 ℃ for 30 min; 94 ℃ for 2 min; then the temperature is 94 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 1 min; and 30 cycles. Extension at 72 ℃ for 10 min. Recovering PCR products by electrophoresis gel, connecting with a pMD19-T vector, transforming DH5 alpha competent cells, screening positive colonies on an ampicillin resistant LB plate, extracting plasmids, and digesting pMD19-T-HA/M/NA plasmid restriction enzyme identification respectively by Xho I/EcoRI/Kpn I. Sequencing by positive plasmid committee bioengineering (Shanghai) Co., Ltd, as shown in SEQ ID NO.4, NO.5 and NO. 6.

3. The expression plasmids of avian influenza virus HA, M and NA are constructed to sequence correct pMD19-T-HA/M/NA plasmids, pMD19-T-HA is digested by EcoRV/Not I, pMD19-T-M and pMD19-T-NA are digested by SnaB I and Not I, respectively, the plasmids are connected with p8.5K digested by SnaB I and Not I, DH5 alpha competent cells are transformed, positive colonies are screened and amplified on an ampicillin plate, plasmids are extracted, Not I digestion identification is carried out, positive persons are sequenced by committee biotechnology (Shanghai) limited company, and the plasmids with correct sequences are named as p 8.5K-HA/M/NA.

TABLE 1 structural proteins HA, M1 and NA Gene amplification primers

Note: restriction enzyme recognition sites are underlined.

4. Test results

The sequencing result of the plasmid shows that the homologous arm sequence is consistent with the partial sequence of 18S rDNA (GenBank: FN392325.1) of the GS115 strain of the Pichia pastoris, and the total length is 1292 bp; the length of the multicopy integration vector p8.5K is 8302bp, and after the influenza virus HA, M and NA genes are inserted, Not I enzyme digestion identifies expression plasmids, and the lengths respectively reach 10.0Kb, 9.3Kb and 9.6Kb (figure 1). The sequencing results were also consistent with the target sequence.

Example two: recombinant yeast construction and avian influenza HA, M1 and NA expression

Respectively carrying out amplification culture and extraction on p8.5K-HA/M/NA plasmids, digesting by Kpn21, and carrying out phenol: chloroform: isopentanol (25:24:1) nucleic acid extraction method recovered linearized plasmid, and the concentration of nucleic acid was determined to be 1. mu.g/. mu.L. Then, the p8.5k-HA/M/NA plasmid was mixed in a ratio of HA: M: NA ═ 4:10:1 (v/v). Plasmid electrotransfer was performed according to the pPIC9K A Pichia Vector for Multicopy Integration and Secreted Expression (Catalog No. V175-20) operating manual. p8.5K was also used as a control. The transfer product was then left at room temperature for 1 hour, 1.25mL of BMGY was added, and the mixture was incubated at 25 ℃ under 250 rpm for 24 hours. 100. mu.L of the culture was applied to 1mg/mL, 2mg/mL, and 3mg/mL geneticin G418 YPD plates, and cultured at 25 ℃ for 3 to 5 days. Selecting a single colony to inoculate BMGY culture medium, culturing at 25 ℃ at 250 rpm for 2-3 days, respectively taking 500 mu L, centrifuging to remove supernatant, and extracting a yeast genome as a PCR template by a helicase-protease K-SDS method. The primers for identification are referred to table 1, and the reaction procedure is: 94 ℃ for 2 min; 30 seconds at 94 ℃; 15 seconds at 55 ℃; 60 seconds at 72 ℃; for a total of 25 cycles. Extension at 72 ℃ for 10 min. The positive recombinant strains should respectively amplify HA/M/NA bands, and the negative strains have no corresponding bands. The positive recombinant bacteria are named rAI-x (x represents the serial number of the strain).

1. Recombinant expression of influenza HA, M1 and NA

The recombinant strain identified as positive by the PCR method is inoculated with BMGY for culture and cultured for 3 days at 25 ℃ under the condition of 250 revolutions per minute. 10mL of the bacterial liquid is centrifuged to collect cells, BMMY culture medium is added to continue the culture, the cells are cultured for 5 days at 25 ℃ under the condition of 250 revolutions per minute, and 0.5 percent methanol is supplemented to the cells on the 2 nd day and the 4 th day respectively. Collecting thallus, washing twice with PBS, adding 0.1% fungal protease inhibitor, and crushing yeast cells by high pressure homogenization at 1700 bar; and repeating the steps once. And repeatedly freezing and thawing for three times, and placing the mixture in a refrigerator at the temperature of 20 ℃ below zero for later use. Taking 10mL of broken yeast cells, centrifuging the supernatant at 20,000g for 30min, and taking the supernatant; after ultracentrifugation at 100,000g for 3 hours, the supernatant was discarded, and the precipitate was dissolved in STE buffer to 1 mL. The protein content of the samples was determined according to the modified Bradford method and the dilution was quantified at 1. mu.g/. mu.L.

A2.0 mL centrifuge tube (matched with an MLA-150Fixed Angle rotator) of Quick-seal polyallomer, bell top is taken, 571.4 mu L of 60% sucrose solution, 571.4 mu L of 45% sucrose solution, 571.4 mu L of 30% sucrose solution and 285.7 mu L of STE-dissolved protein precipitate are sequentially added into each tube. When the sample is added, the sample is injected along the tube wall by an injector lightly so that the sample slides down along the wall to prevent the liquid level from being damaged. The electric heating probe heats the centrifugal pipe orifice and seals. Centrifuging at 100,000g for 3h, sucking protein layer with long needle, collecting, and diluting with STE buffer solution. Freezing and storing at the temperature of minus 20 ℃, measuring the content by a spectrophotometer, and concentrating and quantifying to 100 mu g/mL.

2. Identification of influenza viruses HA, M1 and NA

Taking the supernatant of the crushed recombinant yeast cell, 45% sucrose layered protein (protein is not collected in 30% and 60% sucrose layered), inactivated avian influenza virus, the supernatant of the crushed negative control yeast, the supernatant of the crushed blank yeast cell, and the like, and adding the same amount of 2 xSDSMixing the sample buffer solutions, boiling at 100 ℃ for 10min, and carrying out SDS-PAGE electrophoresis; and dyeing and decoloring the electrophoresis gel. Transferring the protein to a PVDF membrane by the other piece of electrophoresis gel through a wet electrotransformation method; constant pressure 100V 60min, rinsing with water, adding 0.5% skimmed milk PBS (Na)₂HPO₄，8mM；NaCl，136mM；KH₂PO₄2 mM; KCl, 2.6mM), and sealing for 2 hours at room temperature by low-speed shaking; PBST (PBS, Tween-20, 0.05% (v/v)) washing membrane three times; adding 1: 50 diluted inactivated avian influenza virus immune chicken serum (purchased from national reference laboratories for avian influenza) is subjected to low-speed shaking at room temperature for 2 hours; PBST membrane washing three times; adding HRP-rabbit anti-chicken IgY (diluted at a ratio of 1: 1000), and shaking at room temperature at low speed for 2 h; PBST membrane washing for three times, and putting a DAB color development solution (0.01mol/L Tris-HCl, pH7.6; 0.05% 3, 3-tetra hydrochloric acid diaminobenzidine) which is prepared freshly; adding 10 μ L of 30% H at the time of application₂O₂The reaction was stopped by rinsing with water after developing for 10 minutes at room temperature in the dark, and the results were observed.

3. Test results

Several single colonies growing on YPD plates of 3mg/mL G418 were picked up, cultured, and genomic DNA was extracted by helicase-proteinase K-SDS method while negative and blank controls were set up for PCR amplification of HA/M/NA genes. The results showed that there were 2 recombinant yeast genomes with HA/M/NA target bands and negative and blank controls without corresponding bands (FIG. 2). Positive recombinant strains were designated rAI-a/1 and rAI-a/2.

Amplification culture of rAI-a/1 and rAI-a/2, SDS-PAGE electrophoresis of thalli broken supernatant shows that a recombinant strain protein band and negative and blank controls have a difference band (figure 3), but the recovery rate is low, and only 45% sucrose layered protein is collected; western-blot atlas shows that the recombinant strain rAI-a/1 has three bands of 64KD, 49KD and 28KD which are completely different from negative and blank control strains; meanwhile, the virus control was established (fig. 4). The test result shows that the HA, M and NA protein expression quantity of the recombinant strain rAI-a/1 is relatively balanced.

Example three: recombinant avian influenza virus-like particle immunoassay

1. Extracting, purifying and quantifying recombinant HA, M1 and NA

The positive recombinant strain rAI-a/1 is inoculated with BMGY for culture and cultured for 3 days at 25 ℃ under the condition of 250 revolutions per minute. Collecting cells by centrifugation of 100mL of bacterial liquid, adding BMMY culture medium, continuing culturing at 25 ℃ under 250 rpm for 5 days, and supplementing 0.5% methanol at 2 days and 4 days respectively. Collecting thallus, washing twice with PBS, adding 0.1% fungal protease inhibitor, and crushing yeast cells by high pressure homogenization at 1700 bar; and repeating the steps once. And repeatedly freezing and thawing for three times. 100mL of the disrupted yeast cells were collected, centrifuged at 12,000g for 10min, and the supernatant was collected. 100mL of supernatant was added with solid ammonium sulfate with slow stirring to a final concentration of 10%, allowed to stand at 4 ℃ for 6 hours, centrifuged at 12,000g for 10min, and the supernatant was collected. Solid ammonium sulfate was slowly added to the supernatant with stirring to a final concentration of 25%, and the mixture was allowed to stand at 4 ℃ for precipitation overnight. The pellet was harvested by centrifugation at 12,000g for 30min at 4 ℃. Desalting by dialysis at 4 deg.C, concentrating with PEG20000, and quantifying to 1 μ g/μ L.

After SDS-PAGE electrophoresis gel is dyed and decolored, ImageJ software is adopted to determine the relative amount of each band; the positive bands of the western-blot verification correspond to SDS-PAGE electrophoresis gel; the protein content of the samples was determined according to the modified Bradford method. And finally, according to a formula: HA. The total amount of M1 and NA protein, i.e. the sample protein amount × the target protein purity factor × 100%, and the amounts of HA, M1 and NA protein, which add up to the crude VLPs, were determined.

Preparing an electron microscope sample from the purified protein, negatively dyeing with phosphotungstic acid, adsorbing on a copper mesh, and observing under an H-600 transmission electron microscope.

2. Immunological test

100 mu g/mL of recombinant protein is taken and added with the same amount of MONTANIDE ISA 206VG adjuvant, and the mixture is fully emulsified and mixed evenly. Selecting 21-28 days old SPF chicks, and randomly dividing 30 SPF chicks into 6 groups. Wherein, the oil adjuvant recombinant virus-like particles are immunized into three groups of 5 groups, each group is respectively 100 muL/one, 200 muL/one and 400 muL/one, and chest intramuscular injection is carried out; 5 positive control groups, 500 mu L of inactivated avian influenza vaccine and breast intramuscular injection; negative control group 5, oil adjuvant recombinant empty carrier yeast protein, 500 u L/one, chest intramuscular injection; the control group contained 5 mice, PBS mixed with an equal amount of adjuvant, 500. mu.L/mouse, injected intramuscularly in the chest. The groups and doses are shown in table 2. Whole blood was collected at 0, 1, 3, 6, and 10 weeks after immunization, serum was separated, and serum in each group was mixed, and Hemagglutination (HA) and hemagglutination inhibition assay (HI) were performed to detect antibody titers [ refer to highly pathogenic avian influenza diagnosis technology (GB/T18936-2003) ].

TABLE 2 immunoassay groupings and dosages

3. Test results

3.1 Virus-like particle morphology and concentration

The relative amounts of HA, M1 and NA bands were determined by ImageJ software using recombinant protein at a concentration of 1. mu.g/. mu.L, and the virus-like particle content was determined to be 0.10. + -. 0.02. mu.g/. mu.L. The electron microscope picture shows that the virus-like particles are complete and clear, the diameter is about 80-120 nm, and the shape of the virus-like particles is consistent with that of the avian influenza virus particles (figure 5).

3.2 Immunity antibody

The recombinant protein 100 mu L/chick immune group 1, the blood coagulation inhibition price at 0, 1, 3, 6 and 10 weeks of serum are respectively:<2^-1、2^-2、2^-5、2^-4、2^-2(ii) a The recombinant protein 200. mu.L/chick immune group 2, the blood coagulation inhibition price at 0, 1, 3, 6 and 10 weeks of serum are respectively:<2^-1、2^-3、2^-6、2^-5、2^-3(ii) a Recombinant protein 400. mu.L/chick immune group 3, serum hemagglutination inhibition values at 0, 1, 3, 6 and 10 weeks are respectively:<2^-1、2^-3、2^-6、2^-6、2^-4(ii) a The hemagglutination inhibition valence of the whole virus inactivated vaccine is respectively<2^-1、2^-4、2^-8、2^-8、2^-6. The level of hemagglutination antibody of the chick immunized by the PBS and the empty vector recombinant yeast protein is always<2^-1. The test result shows that the hemagglutination inhibition price of the recombinant protein immune group is slightly lower than that of the whole virus inactivated vaccine group, but the difference with the control group is extremely obvious, and the recombinant protein immune group has the potential to be used as a gene engineering virus-like particle vaccine for the immunization of avian influenza.

Sequence listing

<110> veterinary institute of the department of veterinary sciences, Sinkiang (animal clinical medicine research center of the department of veterinary sciences, Sinkiang)

<120> avian influenza virus-like particle assembly expression system and application thereof

<130> 12

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1292

<212> DNA

<213> target sequence of integration region of Pichia pastoris foreign Gene multicopy ()

<400> 1

tgtacaaagg gcagggacgt aatcagcgcg agctgatgac tcgcgcttac taggaattcc 60

tcgttgaagc gcctcttgca aagcgctatc cccagcacga cggagtctaa gattccccgg 120

ccatctctgg caaggactcg ctgcctccgt cagtgtagcg cgcgtgcggc ccagaacgtc 180

taagggcatc acagacctgt tattgcctcg cttccgctgg cttgcgccag ttgtccttct 240

aagaagatcc cccagcaatg ccaggtaacc tagttaaaag ccaaggtctc gttcgttatc 300

gcaattaagc agacaaatca ctccaccaac taagaacggc catgcaccac cacccacaaa 360

atcaagaaag tgctctcatc ctgtcaatcc tcattgtgtc tggacctggt gagtttcccc 420

gtgttgagtc aaattaagcc gcaggctcca ctcctggtgg tgcccttccg tcaattcctt 480

taagtttcag ccttgcgacc atactccccc cagaacccaa agactttgat ttctcgtaag 540

gtgccgggga aggctattcc ccgatcccta gtcggcatcg tttatggtta agactacgac 600

ggtatctgat catcttcgat cccctaactt tcgttcttga ttaatgaaaa cgtccttggc 660

gaatgctttc gcagtagtta gtcttggggc gatccaagaa tttcacctct gacgccccaa 720

tactgacgcc cccgaccgtc cctgttaatc attacgcggc cccgaaccaa caaaagaacc 780

gtatcctctt ctgttattcc atgctaatat attcaactac tgccttgaac actctaattt 840

cctcaaagta acgtccgttc aactacgagc tttttaactg caacaacttt aatatacgct 900

attggagctg gaattaccgc ggctgctggc accagacttg ccctccaatt gttcctcgtt 960

aaggtattta cgttgtactc attccaatta caagaccaaa ggccctgtat cgttatttat 1020

tgtcactacc tccctgtgtc aggattgggt aatttgcgcg cctgctgcct tccttggatg 1080

tggtagccgt ctctcaggct ccctctccgg aatcgaaccc ttattccccg ttacccgtag 1140

aaaccatggt aggcctctat cctaccatcg aaagttgata gggcagaaat ttgaatgaac 1200

catcctaaga ttcgaaaagt tattatgaat caccaaaacg aaggttttat ctaataaata 1260

cgcccgaggg ctgatcaagt attagctcta ga 1292

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 2

tggttcattc aaattcaaat ttct 24

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 3

ccagaaccca aaaactttga t 21

<210> 4

<211> 1704

<212> DNA

<213> HA Gene sequence in avian influenza Virus-like particle expression plasmid p8.5K-HA ()

<400> 4

atggagaaaa tagtgcttct tcttgcaaca atcagtcttg ttaaaagtga tcagatttgc 60

attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120

actgttacac atgctcaaga catactggaa aagacacaca acgggaagct ctgcgaccta 180

gatggagtga agcctctaat tttgagagat tgtagtgtag ctggatggct cctcggaaac 240

ccaatgtgtg acgaattcat caatgtgccg gagtggtctt acatagtgga gaaggccagt 300

ccagccaatg acctctgtta cccaggggat ttcaacgact atgaagaact gaaacaccta 360

ttgagcagaa taaaccattt tgagaaaatt cagatcatcc ccgaaagttc ttgggccaat 420

catgaagcct catcaggggt gagctcagca tgtccctacc tggggaagcc ctcctttttc 480

agaaatgtgg tatggcttat caaaaagaat aatacgtacc caacaataaa gaggagctac 540

aataatacca accaagaaga tcttttggta ctgtggggga ttcaccatcc taatgatgcg 600

gagcagataa agctctatca aaacccaacc acctatatct ccgttggaac atcaacacta 660

aaccagagat tggtaccaaa aatagctact agatccaaag taaacgggca aagtggaaga 720

atggagttct tctggacaat tttaaagccg aatgatgcta tcaatttcga gagtaatgga 780

aatttcattg ttccagaata tgcatacaaa attgtcaaga aaggggactc tgcaattatg 840

agaagtgaat tggaatatgg taactgcaac accaagtgtc aaactccaat gggggcgata 900

aattctagta tgccattcca caacatacac cctctcacca tcgaggaatg ccccaaatat 960

gtgaaatcaa acagattagt ccttgcgact ggactcagaa atgcccctca aagagaggga 1020

agaagaaaaa agagaggact atttggagct atagcagggt ttatagaggg aggatggcag 1080

ggaatggtag atggttggta tgggtaccac catagcaatg agcaggggag tggatacgct 1140

gcagacaaag aatccactca aaaggcaata gatggagtca ccaataaggt caactcgatc 1200

attgacaaaa tgaacactca gtttgaggcc gttggaaggg aatttaataa cttagaaagg 1260

agaatagaaa atttaaacaa gaagatggag gacggattcc tagatgtctg gacttataat 1320

gctgaacttc tggttctcat ggaaaatgag agaactctgg actttcatga ctcaaatgtc 1380

aagaaccttt acgaaaaggt ccgactacag cttagggata atgcaaagga gttgggtaac 1440

ggttgtttcg agttctatca caaatgtgat aatgaatgta tggaaagtgt aaaaaacgga 1500

acgtatgact acccgcagta ttcagaagaa gcaagactaa acagagagga aataagtgga 1560

gtaaaattgg aatcaatggg aacttaccaa atactgtcaa tttattcaac agtggcgagt 1620

tccctagcac tggcaatcat ggtagctggt ctatctttat ggatgtgctc caatggatcg 1680

ttacaatgca gaatttgcat ttaa 1704

<210> 5

<211> 982

<212> DNA

<213> expression of M Gene sequence in plasmid p8.5K-M for avian influenza Virus-like particles ()

<400> 5

atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccgtc aggccccctc 60

aaagccgaga tcgcgcagaa acttgaagat gtcttcgcag gaaagaacac cgatctcgag 120

gctctcatgg agtggctaaa gacaagacca atcctgtcac ctctgactaa agggattttg 180

ggatttgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240

cagaatgccc taaatggaaa tggagatcca aatgatatgg atagggcagt taagctgtat 300

aagaagctga aaagagaaat aacattccat ggggctaagg aggtcgcact cagctactca 360

accggtgtac ttgccagttg catgggtctc atatacaaca ggatgggaac ggtgactacg 420

gaagtggctt ttggcttagt gtgtgccact tgtgagcagg ttgcagattc acagcatcgg 480

tctcacagac agatggcaac tatcaccaac ccactaatca ggcatgagaa cagaatggtg 540

ctggccagca ctacagctaa ggctatggag cagatggcgg gatcaagtga gcaggcagcg 600

gaagccatgg aggtcgctaa tcaggctagg cagatggtgc aggcaatgag gacaattggg 660

actcatccta actctagtgc tggtctgaga gataatcttc ttgaaaattt gcaggcctac 720

cagaaacgaa tgggagtgca gatgcagcga ttcaagtgat cctcttgttg ttgccgcaag 780

tatcattggg gtcttgcact tgatattgtg gattcttgat cgtcttttct tcaaatgcat 840

ttatcgtcgc cttaaatacg gtttgaaaag agggccttct acggcagggg tacctgagtc 900

tatgagggaa gagtaccggc aggaacagca gaatgctgtg gatgttgacg atggtcattt 960

tgtcaacata gaattggagt aa 982

<210> 6

<211> 1350

<212> DNA

<213> NA gene sequence in expression plasmid p8.5K-NA of avian influenza virus-like particle ()

<400> 6

atgaatccaa atcagaagat aataaccatc ggatcaatct gtatggtaat tggaatagtt 60

agcttaatgt tacaaattgg gaacatgatc tcaatatggg tcagtcattc aattcagaca 120

gggaatcaac gccaagctga accgatcagc aatactaaat ttcttactga gaaagctgtg 180

gcttcagtaa cattagcggg caattcatct ctttgcccca ttagcggatg ggctgtacac 240

agtaaggaca acagcataag gatcggttcc aagggggatg tgtttgttat aagagagccg 300

ttcatctcat gctcccactt ggaatgcaga actttctttt tgactcaggg agccttgctg 360

aatgacaagc actccaatgg gactgtcaaa gacagaagcc cccacagaac attaatgagt 420

tgtcctgtgg gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca 480

gcaagtgctt gccatgatgg caccagttgg ttgacaattg gaatttctgg cccagacaat 540

ggggctgtgg ctgtattgaa atacaatggc ataataacag acactatcaa gagttggagg 600

aacaacatac tgagaactca agaatctgaa tgtgcatgtg taaatggctc ttgctttact 660

gtaatgactg atggaccaag taatgggcag gcatcatata agatcttcaa aatggaaaaa 720

gggaaagtgg ttaaatcagt cgaattggat gctcctaatt atcactatga ggagtgctcc 780

tgttatcctg atgccggcga aatcacatgc gtgtgcgggg ataattggca cggctcaaat 840

aggccatggg tatctttcaa tcaaaattta gagtatcaaa taggatatat atgcagtgga 900

gttttcggag acaatccacg ccccaatgat ggaacaggta gtcgtggtcc ggtgttccct 960

aacggggcat atggggtaaa agggttttca tttaaatacg gcaatggtgt ttggatcggg 1020

agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg 1080

actggaacgg acagcagctt ttcggtgaag caagatatcg tagcaataac tgattggtca 1140

ggatatagcg ggagttttgt ccagcatcca gaactgacag gattagattg cataggacct 1200

tgtttctggg ttgagttagt cagagggcgg cccaaagaga gcacaatctg gactagtggg 1260

agcagcatat ctttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1320

gctgagttgc cattcaccat tgacaagtag 1350

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 7

aaaatagtgc ttcttcttgc a 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 8

ttaaatgcaa attctgcatt g 21

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 9

agtcttctaa ccgaggtcga a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 10

ttactccaat tctatgttga c 21

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 11

aatccaaatc agaagata 18

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 12

ctacttgtca atggtgaa 18

Claims (8)

1. Pichia pastoris GS115/rAI-a/1 (A/A) for recombinant expression of avian influenza H5N1 virus-like particlespichia pastoris strain GS115/rAI-a/1) The preservation number of the recombinant strain is CCTCC No: m2020630.

2. A target sequence of a pichia pastoris exogenous gene multicopy integration region is located in the middle of an 18S rDNA gene and is shown in SEQ ID NO. 1.

3. Designing a primer pair according to the target sequence in claim 2, and obtaining the sequence by PCR amplification for constructing a Pichia pastoris multicopy integration vector p8.5K; in the primer pair, the upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO. 3.

4. The Pichia pastoris multi-copy integration vector p8.5K of claim 3, by insertion of SEQ ID No.1 into eukaryotic expression vector pPIC9KXbaI andBsp1047Ⅰrestriction sites.

5. An avian influenza virus-like particle expression plasmid p8.5K-HA, p8.5K-M and p8.5K-NA constructed by a multi-copy integration vector p8.5K, wherein nucleic acid sequences of hemagglutinin HA genes and M genes, namely including matrix protein M1, ion channel protein M2 and neuraminidase NA genes, are amplified from an avian influenza virus H5N1 genome by an RT-PCR method and respectively correspond to gene sequences shown as SEQ ID No.4, No.5 and No. 6.

6. The expression plasmid p8.5K-HA according to claim 5, which is prepared byEcoRV andNot Irestriction enzyme digestion separately, insertion into multicopy integration vector p8.5KSnaBI andNot Iobtained between the cleavage sites.

7. The expression plasmids p8.5K-M and p8.5K-NA according to claim 5, respectively, are prepared bySnaBI andNot Irestriction enzyme digestion, insertion into the multicopy integration vector p8.5KSnaBI andNot i is obtained between enzyme cutting sites.

8. The HA, M and NA gene according to claim 5, wherein the three primer pairs amplified are: (1) an upstream primer SEQ ID NO.7 and a downstream primer SEQ ID NO. 8; (2) an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO. 10; (3) an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO. 12.

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