CN111285924A

CN111285924A - Flic immune adjuvant based on baculovirus expression system, preparation method and application

Info

Publication number: CN111285924A
Application number: CN202010092764.3A
Authority: CN
Inventors: 樊惠英; 王亚贞; 孔德鑫; 胡朝升; 何恒昌
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-02-14
Filing date: 2020-02-14
Publication date: 2020-06-16

Abstract

The invention discloses a Flic immune adjuvant based on a baculovirus expression system, a preparation method and application thereof. The invention expresses and purifies the nucleotide sequence of encoding salmonella typhimurium flagellin through a Bac-to-Bac baculovirus expression system, and the obtained soluble Flic protein is Flic immunological adjuvant. The soluble Flic protein obtained by the method provided by the invention has high yield, and has extremely high physicochemical property, biological activity, immunogenicity and safety. The inactivated avian influenza vaccine prepared by the Flic immune adjuvant can quickly generate HI antibody titer with high titer after immunization, can induce generation of cellular immune response, and has good immunogenicity and reactivity.

Description

Flic immune adjuvant based on baculovirus expression system, preparation method and application

Technical Field

The invention belongs to the field of biological products, and particularly relates to a Flic immune adjuvant based on a baculovirus expression system, and a preparation method and application thereof.

Background

Adjuvants are substances that are injected into poultry prior to or simultaneously with an antigen, and that can accelerate, prolong or enhance the body's specific immune response to the antigen or alter the type of immune response, but are not immunogenic themselves and cannot elicit the body's immune response. As a unique bacterial pathogen-associated molecular pattern (pathogen-associated molecular pattern, PAMPs), flagellin has the immunological activity of activating two signal pathways of Toll-like receptor 5(TLR5) and NLR family (NLRC4), and can generate wide natural immune response and acquired immune response to specific antigens, thereby effectively activating the cellular and humoral immune response of the body.

At present, an escherichia coli expression vector is often used as a common vector for expressing target protein due to the characteristics of easy culture and high expression quantity, but an expression system of the escherichia coli expression vector does not have a complete post-translational processing modification function, so that the biological activity of an expression product is low, and the expression product often exists in an inclusion body form, so that the product is difficult to purify.

Therefore, it is necessary to develop a more active immunoadjuvant.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation method of a Flic immune adjuvant based on a baculovirus expression system.

Another object of the present invention is to provide a Flic immunoadjuvant obtained by the above-mentioned preparation method.

The invention further aims to provide application of the Flic immune adjuvant.

The purpose of the invention is realized by the following technical scheme: a preparation method of Flic immune adjuvant based on baculovirus expression system comprises the following steps: expressing and purifying a nucleotide sequence for coding salmonella typhimurium flagellin by a Bac-to-Bac baculovirus expression system to obtain soluble Flic protein; preferably comprising the steps of:

(1) cloning a nucleotide sequence for coding flagellin of salmonella typhimurium into a transfer plasmid to obtain a recombinant transfer plasmid;

(2) transposing and recombining the recombinant transfer plasmid obtained in the step (1) through Tn7, transposing and recombining a nucleotide sequence for coding salmonella typhimurium flagellin to a baculovirus shuttle vector Bacmid to obtain a recombinant baculovirus plasmid;

(3) transfecting the recombinant baculovirus plasmid obtained in the step (2) into insect host cells, and collecting supernatant to obtain first-generation baculovirus;

(4) carrying out amplification culture on the first generation baculovirus;

(5) infecting insect host cells with the first generation baculovirus or baculovirus obtained by amplification culture;

(6) collecting cells, and purifying to obtain soluble Flic protein which is a Flic immune adjuvant based on a baculovirus expression system.

The amino acid sequence of the salmonella typhimurium flagellin comprises a sequence shown as SEQ ID NO. 1; preferably, a 6 × HIS tag is added to the N-terminus and/or C-terminus of the sequence shown in SEQ ID NO.1 to facilitate later purification.

The nucleotide sequence for coding the flagellin of the salmonella typhimurium comprises a sequence shown as SEQ ID NO. 3; preferably as shown in SEQ ID NO. 4.

The transfer plasmid is preferably pFastBac ^TM1, the nucleotide sequence coding the flagellin of the salmonella typhimurium is constructed in the P of the transfer plasmid_HDownstream of the promoter. Thus, the expression of Flic gene is by Autographa californica Polykaryotic polyhedrosis Virus (AcMNPV) polyhedron (P)_H) The control of the promoter is beneficial to the high-level expression of the Flic gene in insect cells.

The Tn7 transposition recombination in the step (2) is realized by the following steps: transforming the recombinant transfer plasmid into a DH10Bac escherichia coli competent cell, transposing and recombining through Tn7, transposing and recombining a nucleotide sequence for coding salmonella typhimurium flagellin to a baculovirus shuttle vector Bacmid to obtain a recombinant baculovirus plasmid.

The transfection in step (3) is preferably a lipofection.

The insect host cell described in step (3) is preferably an Sf9 cell.

The expanding culture in the step (4) means that the first generation of baculovirus is infected into insect host cells, the supernatant is baculovirus, and the step of continuously infecting the obtained virus into the insect host cells can be repeated according to actual requirements to obtain a larger amount of baculovirus; preferably comprises the following specific steps: the first generation of recombinant baculovirus was infected at a cell density of 2 × 10 at an MOI of 0.1⁶And (3) carrying out suspension culture on sf9 cells per ml at the temperature of 27-28 ℃ for 72h, and collecting cell culture supernatant, namely the second-generation recombinant baculovirus.

The temperature of the suspension culture is preferably 27 ℃.

Step (5) is preferably as follows: infecting the second generation recombinant baculovirus with MOI of 1-5 to obtain 2.5 × 10 cell density⁶The cells were collected after suspension culture at 27-28 ℃ for 72h per ml of sf9 cells.

The temperature of the suspension culture is preferably 27 ℃.

The specific steps of purification described in step (6) are as follows: and (3) carrying out ultrasonic treatment on the collected cells, carrying out solid-liquid separation on the treatment liquid, and purifying the target protein with the His label by using Ni-NAT affinity chromatography on the obtained supernatant to obtain the soluble Flic protein.

The conditions for the ultrasonic treatment are preferably as follows: the working time is 3s, the intermittent time is 5s, the working power is 100-200 w, and the crushing time is 10-15 min.

The solid-liquid separation method is preferably centrifugation.

The centrifugation condition is preferably 4 ℃ and 10000rpm for 8-10 min.

The steps of the Ni-NAT affinity chromatography are preferably as follows: and (3) putting the supernatant on a Ni-NAT column, fully mixing the supernatant and Ni uniformly, standing, eluting the hybrid protein by using 100mM imidazole after the unbound protein liquid flows out of the Ni-NAT column through the action of gravity, and directly eluting the target protein with His by using 200-500 mM imidazole to obtain the soluble Flic protein.

The molecular weight of the soluble Flic protein is about 53.6 KDa.

A Flic immunoadjuvant based on a baculovirus expression system, obtained by the above preparation method, has physicochemical and biological properties similar to those of a natural protein.

The application of the Flic immune adjuvant based on the baculovirus expression system in preparing vaccines preferably comprises the following steps: mixing the Flic immune adjuvant based on the baculovirus expression system with inactivated virus to serve as a vaccine water phase; and mixing the vaccine aqueous phase with a water-in-oil-in-water adjuvant to obtain the vaccine.

The virus is preferably avian influenza virus; more preferably H9N2 avian influenza virus.

The water-in-oil-in-water adjuvant is preferably MONTANIDE ISA series adjuvant; more preferably, the adjuvant is MONTANIDE ISA201 VG.

The components of the vaccine are preferably as follows: 50% volume of water-in-oil-in-water adjuvant, 20 mug/0.3 ml of Flic immune adjuvant based on baculovirus expression system, and not less than 5 x 10 of inactivated virus content before virus inactivation per 0.1ml⁷EID 50。

Compared with the prior art, the invention has the following advantages and effects:

1. the invention optimizes the nucleotide sequence according to the species of expressing the target gene, namely Spodoptera frugiperda, and realizes the stable and large-scale expression of the target protein.

2. The salmonella typhimurium flagellin Flic-B is expressed by a Bac-to-Bac baculovirus expression system, and the expression system belongs to a eukaryotic expression system, so that an expression product and a natural exogenous product have extremely high physicochemical property, biological activity and immunogenicity, and the expression product has higher biological safety because the expression product has no infection to vertebrates.

3. According to the invention, through designing a primer, 6-His tags are respectively inserted into the upstream and the downstream of the primer, and the His tags are successfully inserted into the N end and the C end of a target gene through PCR amplification, so that the expression identification and purification of a target protein are facilitated.

4. In the embodiment of the invention, Flic-B protein is expressed by using a mode of shake flask suspension culture of insect cells, a large amount of single target protein can be purified by using 20ml of shake flask culture cells, and Flic-B protein can be expressed in a large amount by using a fermentation tank and the like in the future, so that the production cost of the adjuvant can be greatly reduced.

5. Compared with Flic-E protein induced and expressed by escherichia coli, the Flic-B protein expressed by the shake flask suspension culture method is higher in biological activity and better in expression amount.

5. The inactivated avian influenza vaccine containing the immune adjuvant Flic provided by the invention can quickly generate HI antibody titer with high titer after immunization, can induce generation of cellular immune response, and has good immunogenicity and reactivity.

Drawings

FIG. 1 is a graph showing the amplification results of a target gene; among them, lanes 1-4 are Flic amplified bands, lane 5 is a negative control, and lane M is DL2000 marker.

FIG. 2 is a diagram showing the results of enzyme digestion identification of the recombinant plasmid pFastBacTM 1-flash; wherein, lane 1 is a DNA fragment obtained by double digestion of plasmid pFastBacTM 1-flash with BamH I and Hind III, and lane M is DL5000 marker.

FIG. 3 is a diagram showing the result of identification of the recombinant baculovirus plasmid Bacmid-Flic; wherein, lane 1 is the PCR result of primer amplification of M13, lane 2 is the result of Flic primer amplification, lane M1 is DL2000 marker, and lane M2 is DL5000 marker.

FIG. 4 is a graph showing the result of IFA identification of Flic-B protein expression; wherein, a is a normal cell map, and b is a result map of BV-Flic infected Sf9 cells.

FIG. 5 is a graph showing the results of SDS-PAGE analyzing the expression of Flic-B protein at different MOIs; wherein,

lanes

1, 2 and 3 are supernatants obtained by ultrasonic treatment of Flic-B protein expressed when MOI is 1, 3 and 5,

lanes

4, 5 and 6 are precipitates obtained by ultrasonic treatment of Flic-B protein expressed when MOI is 1, 3 and 5, lane 7 is wild virus control, and lane M is protein maker (size 180 KDa).

FIG. 6 is a graph showing the expression results of Flic-B protein under different MOIs in Western-Blot analysis; wherein,

lanes

FIG. 7 is a diagram showing the results of SDS-PAGE analysis for purification of Flic-B protein; wherein, Lane 1 is the original protein solution, Lane 2 is the protein not bound to the nickel column, Lane 3-9 are the purified proteins eluted with 20mM, 50mM, 100mM, 200mM, 300mM, 400mM, 500mM imidazole, respectively, and Lane M is the protein maker (size 170 KDa).

FIG. 8 is a graph showing the results of purification of Flic-B protein for adjuvant preparation; wherein, lane 1 is the expressed original protein solution, lane 2 is the protein not bound to the nickel column, lane 3 is the hybrid protein eluted by 100mM imidazole, lanes 4-5 are the target protein Flic-B with a single band eluted by 200mM imidazole, lane 6 is the target protein Flic-B with a single band eluted by 500mM imidazole, and lane M is the protein maker (size 170 KDa).

FIG. 9 is a diagram showing the results of restriction enzyme identification of recombinant plasmid pET-32 a-Flic; wherein, lane 1 is a DNA fragment obtained by double digestion of plasmid pET-32a-Flic with BamH I and HindIII, and lane M is DL5000 marker.

FIG. 10 is a diagram showing the results of expression identification of Flic-E protein by SDS-PAGE analysis; wherein, Lane 1 is a negative control,

Lane

2, 3, 4 are supernatants of expressed Flic-E protein after sonication, and Lane M is a protein maker (size 180 KDa).

FIG. 11 is a graph showing the results of identifying the expression of Flic-E protein by Western-Blot analysis; wherein, Lane 1 is a negative control,

Lane

FIG. 12 is a graph showing the results of SDS-PAGE analysis of the expression of Flic-E protein induced at different temperatures; wherein,

lanes

1, 2 and 3 are respectively Flic-E protein induced at 27 deg.C, 30 deg.C and 37 deg.C, and lane M is protein maker (size 180 KDa).

FIG. 13 is a diagram showing the results of SDS-PAGE analysis of purification of Flic-E protein for adjuvant preparation; wherein lane 1 is the original protein solution, lane 2 is the protein not bound to the nickel column, lane 3 is the hybrid protein eluted with 100mM imidazole, lanes 4-5 are the target protein Flic-E eluted with 200mM imidazole, lane 6 is the protein eluted with 500mM imidazole, and lane M is the protein maker (size 170 KDa).

FIG. 14 is a graph showing the results of analysis of HI antibody titers at different time points in each immunization group.

FIG. 15 is an analysis chart of the results of cellular immune responses of each immune group after immunization.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test materials in the following examples were all commercially available unless otherwise specified. The test methods are conventional test methods unless otherwise specified.

Example 1 construction and expression of recombinant plasmid of Salmonella typhimurium flagellin Flic-B based on baculovirus expression System

1.1 Synthesis and amplification of Salmonella typhimurium flagellin Flic Gene

Nucleotide optimization is carried out according to Flic gene of a reference strain intestinal serotype salmonella typhimurium genome sequence (CP001363) logged on GenBank, the Flic gene is synthesized, the nucleic acid sequence of the reference gene is shown as SEQ ID NO.2, the nucleic acid sequence of the optimized Flic gene is shown as SEQ ID NO.3, a primer is designed according to the nucleic acid sequence of the optimized Flic gene, wherein BamH I and Hind III enzyme cutting sites and 6 XHIS labels are respectively introduced into the upstream and downstream of P1 and P2, and the underlined part is the enzyme cutting site. The sequences involved are as follows:

protein sequence (also SEQ ID NO. 1):

AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR；

the original nucleotide sequence (also SEQ ID NO. 2):

ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCCGCTCTGGGCACCGCTATCGAGCGTCTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGATACGCTGAATGTGCAACAAAAATATAAGGTCAGCGATACGGCTGCAACTGTTACAGGATATGCCGATACTACGATTGCTTTAGACAATAGTACTTTTAAAGCCTCGGCTACTGGTCTTGGTGGTACTGACCAGAAAATTGATGGCGATTTAAAATTTGATGATACGACTGGAAAATATTACGCCAAAGTTACCGTTACGGGGGGAACTGGTAAAGATGGCTATTATGAAGTTTCCGTTGATAAGACGAACGGTGAGGTGACTCTTGCTGGCGGTGCGACTTCCCCGCTTACAGGTGGACTACCTGCGACAGCAACTGAGGATGTGAAAAATGTACAAGTTGCAAATGCTGATTTGACAGAGGCTAAAGCCGCATTGACAGCAGCAGGTGTTACCGGCACAGCATCTGTTGTTAAGATGTCTTATACTGATAATAACGGTAAAACTATTGATGGTGGTTTAGCAGTTAAGGTAGGCGATGATTACTATTCTGCAACTCAAAATAAAGATGGTTCCATAAGTATTAATACTACGAAATACACTGCAGATGACGGTACATCCAAAACTGCACTAAACAAACTGGGTGGCGCAGACGGCAAAACCGAAGTTGTTTCTATTGGTGGTAAAACTTACGCTGCAAGTAAAGCCGAAGGTCACAACTTTAAAGCACAGCCTGATCTGGCGGAAGCGGCTGCTACAACCACCGAAAACCCGCTGCAGAAAATTGATGCTGCTTTGGCACAGGTTGACACGTTACGTTCTGACCTGGGTGCGGTACAGAACCGTTTCAACTCCGCTATTACCAACCTGGGCAACACCGTAAACAACCTGACTTCTGCCCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTAA；

optimized sequence (also SEQ ID NO. 3):

GCTCAGGTCATTAACACTAACTCCCTGAGCCTGCTGACTCAGAACAACCTGAACAAGTCCCAGAGCGCTCTGGGTACCGCTATCGAACGTCTGTCCAGCGGCCTGCGCATCAACTCCGCTAAGGACGACGCTGCTGGCCAGGCCATCGCCAACCGCTTCACCGCTAACATCAAGGGTCTGACCCAGGCCTCCCGCAACGCCAACGACGGTATCTCCATCGCTCAGACCACCGAAGGCGCCCTGAACGAAATCAACAACAACCTGCAGCGCGTGCGCGAGCTGGCCGTCCAATCCGCTAACTCCACTAACAGCCAGAGCGACCTGGACAGCATCCAGGCTGAGATCACCCAGCGCCTGAACGAGATCGACCGCGTGTCCGGTCAGACCCAGTTCAACGGCGTCAAGGTGCTGGCTCAGGACAACACTCTGACTATCCAGGTCGGCGCTAACGACGGCGAGACCATCGACATCGACCTGAAGCAGATCAACAGCCAGACCCTGGGTCTGGACACTCTGAACGTGCAGCAGAAGTACAAGGTGAGCGACACTGCCGCCACCGTCACTGGCTACGCTGACACCACCATCGCCCTGGACAACTCCACTTTCAAGGCTTCCGCCACTGGCCTGGGTGGTACTGACCAGAAGATCGACGGTGACCTGAAGTTCGACGACACCACCGGCAAGTACTACGCCAAGGTCACTGTGACCGGCGGCACTGGCAAGGACGGTTACTACGAGGTCTCCGTGGACAAGACTAACGGTGAAGTGACCCTGGCTGGTGGCGCTACCAGCCCCCTGACTGGTGGCCTGCCTGCCACCGCTACTGAAGACGTGAAGAACGTCCAGGTGGCTAACGCCGACCTGACCGAAGCCAAGGCTGCTCTGACTGCCGCTGGTGTGACTGGTACTGCTTCCGTGGTCAAGATGTCCTACACCGACAACAACGGCAAGACCATCGACGGTGGCCTGGCTGTGAAGGTGGGTGACGACTACTACTCCGCCACCCAGAACAAGGACGGTAGCATCTCCATCAACACTACCAAGTACACCGCCGACGACGGCACCAGCAAGACCGCTCTGAACAAGCTGGGTGGCGCTGACGGCAAGACCGAAGTGGTCTCCATCGGTGGTAAAACCTACGCTGCTAGCAAGGCCGAGGGTCACAACTTCAAGGCTCAGCCTGACCTGGCCGAAGCTGCCGCCACCACCACCGAGAACCCTCTGCAGAAGATCGATGCTGCTCTGGCCCAGGTGGACACTCTGCGCAGCGACCTGGGTGCTGTCCAGAACCGTTTCAACAGCGCCATCACTAACCTGGGTAACACCGTGAACAACCTGACTAGCGCTCGCAGCCGTATCGAGGACTCCGACTACGCCACTGAGGTGTCCAACATGAGCCGCGCTCAGATCCTGCAGCAGGCCGGTACTAGCGTGCTGGCCCAGGCCAACCAGGTCCCCCAGAACGTCCTGAGCCTGCTCCGT；

the sequence actually used for construction (also SEQ ID NO. 4):

ATGCATCACCATCACCATCACGCTCAGGTCATTAACACTAACTCCCTGAGCCTGCTGACTCAGAACAACCTGAACAAGTCCCAGAGCGCTCTGGGTACCGCTATCGAACGTCTGTCCAGCGGCCTGCGCATCAACTCCGCTAAGGACGACGCTGCTGGCCAGGCCATCGCCAACCGCTTCACCGCTAACATCAAGGGTCTGACCCAGGCCTCCCGCAACGCCAACGACGGTATCTCCATCGCTCAGACCACCGAAGGCGCCCTGAACGAAATCAACAACAACCTGCAGCGCGTGCGCGAGCTGGCCGTCCAATCCGCTAACTCCACTAACAGCCAGAGCGACCTGGACAGCATCCAGGCTGAGATCACCCAGCGCCTGAACGAGATCGACCGCGTGTCCGGTCAGACCCAGTTCAACGGCGTCAAGGTGCTGGCTCAGGACAACACTCTGACTATCCAGGTCGGCGCTAACGACGGCGAGACCATCGACATCGACCTGAAGCAGATCAACAGCCAGACCCTGGGTCTGGACACTCTGAACGTGCAGCAGAAGTACAAGGTGAGCGACACTGCCGCCACCGTCACTGGCTACGCTGACACCACCATCGCCCTGGACAACTCCACTTTCAAGGCTTCCGCCACTGGCCTGGGTGGTACTGACCAGAAGATCGACGGTGACCTGAAGTTCGACGACACCACCGGCAAGTACTACGCCAAGGTCACTGTGACCGGCGGCACTGGCAAGGACGGTTACTACGAGGTCTCCGTGGACAAGACTAACGGTGAAGTGACCCTGGCTGGTGGCGCTACCAGCCCCCTGACTGGTGGCCTGCCTGCCACCGCTACTGAAGACGTGAAGAACGTCCAGGTGGCTAACGCCGACCTGACCGAAGCCAAGGCTGCTCTGACTGCCGCTGGTGTGACTGGTACTGCTTCCGTGGTCAAGATGTCCTACACCGACAACAACGGCAAGACCATCGACGGTGGCCTGGCTGTGAAGGTGGGTGACGACTACTACTCCGCCACCCAGAACAAGGACGGTAGCATCTCCATCAACACTACCAAGTACACCGCCGACGACGGCACCAGCAAGACCGCTCTGAACAAGCTGGGTGGCGCTGACGGCAAGACCGAAGTGGTCTCCATCGGTGGTAAAACCTACGCTGCTAGCAAGGCCGAGGGTCACAACTTCAAGGCTCAGCCTGACCTGGCCGAAGCTGCCGCCACCACCACCGAGAACCCTCTGCAGAAGATCGATGCTGCTCTGGCCCAGGTGGACACTCTGCGCAGCGACCTGGGTGCTGTCCAGAACCGTTTCAACAGCGCCATCACTAACCTGGGTAACACCGTGAACAACCTGACTAGCGCTCGCAGCCGTATCGAGGACTCCGACTACGCCACTGAGGTGTCCAACATGAGCCGCGCTCAGATCCTGCAGCAGGCCGGTACTAGCGTGCTGGCCCAGGCCAACCAGGTCCCCCAGAACGTCCTGAGCCTGCTCCGTCATCACCATCACCATCACTAA；

the primer (P1) is arranged on the sequence also shown in SEQ ID NO. 5:

5’-CGGGATCCATGCATCACCATCACCATCACGCTCAGGTCATTAACACTAACTCCC-3’；

the sequence of the downstream primer (P2) is also shown in SEQ ID NO. 6:

5’-CCCAAGCTTTTAGTGATGGTGATGGTGATGACGGAGCAGGCTCAGGACGTTC-3’；

the target gene amplification PCR reaction system is as follows:

PCR reaction procedure: 10s at 98 ℃, 5s at 60 ℃ and 1min at 72 ℃ for 40s for 30 cycles; final extension at 72 ℃ for 2 min. The PCR product was analyzed by agarose gel electrophoresis, and the size of the product was determined, as shown in FIG. 1, lanes 1-4 were the size of the target gene, lane 5 was negative, and the size of the amplified Flic fragment was 1524bp, which is consistent with the expected result.

1.2 recombinant plasmid pFastBac^TMConstruction and characterization of 1-Flic

The PCR product was recovered and digested with BamHI and HindIII, and inserted into the transfer plasmid pFastBac^TM1 (purchased from Invitrogen) and Hind III site, E.coli DH5a competent cells were transformed to obtain the vector pFastBacTM 1-Flic. The recombinant plasmid is identified by double enzyme digestion, and the plasmid is sequenced, so that the result is correct, as shown in FIG. 2, lane M is DNA marker with size of 5000, and lane 1 is the enzyme digestion identification result.

1.3 acquisition and identification of recombinant baculovirus plasmid Bacmid-Flic

The donor plasmid pFastBacTM 1-flash is transformed into DH10Bac escherichia coli competent cells, the recombinant baculovirus plasmid Bacmid-flash is obtained through transposition recombination, and the fragment is amplified by using M13 primer according to the insect baculovirus operation manual of Invitrogen company, so that the result is correct, and is shown in figure 3.

1.4 acquisition and expression identification of recombinant baculovirus BV-Flic

(1) Acquisition of recombinant baculovirus BV-Flic

By using

II Regent (available from Thermo Fisher Scientific Co.) Liposome-mediated transfection method, recombinant baculovirus plasmid Bacmid-Flic transfected sf9 insect cells (available from GIBCO BRL Co.), cultured at 27 ℃ with cells without transfected plasmid as negative control; when the culture is carried out for 72 hours, the cells are diseased, and the cell culture supernatant is collected, thus obtaining the first generation of recombinant baculovirus (P1) BV-Flic.

The method comprises the following specific steps:

sf9 was confirmed to be in logarithmic phase (1.0-2.5X 10)⁶Individual cells/mL) and survival rates above 95%; mixing before use

II Regent liposomes, pipetting 6-8. mu.L into Grace's institute Medium (available from Thermo Fisher scientific Co.),mixing by short vortex; diluting 1 μ L of recombinant baculovirus plasmid to be transfected (the concentration is more than 1000 ng/. mu.L) in 100 μ L of Grace's solution, and gently mixing; mixing the diluted DNA with the diluted DNA

II, mixing (the total volume is about 210 mu L), gently mixing uniformly, and incubating at room temperature for 15-30 min; the mixture in the six well plate was discarded, washed once with Grace's InsectMedium solution and 800. mu.L Grace's InsectMedium solution was pipetted into the liposome mixture to make up to 1 mL; dropwise adding about 1mL of the DNA-liposome mixture or the transfection mixture into the washed cells, and incubating the cells at 27 ℃ for 3-5 h; the mixture was aspirated, washed once with Grace's Instrument Medium solution, and 2mL of complete Medium was added; and (3) culturing the six-hole plate in an incubator at 27 ℃, collecting cell culture supernatant after observing cell lesions, and obtaining the first generation of recombinant baculovirus (P1) BV-Flic.

(2) Identification of expression of Flic-B protein by IFA method

Sf9 cells were plated at 2X 10⁶The individual cells/ml were plated in 6-well plates, according to

The manual of baculovirus expression systems states that it is generally assumed that the titer of the P1 generation recombinant virus is 5X 10⁶pfu/ml, MOI 0.1, sf9 cells were infected with P1 generation recombinant baculovirus for 48h, and then subjected to indirect immunofluorescence assay. The method comprises the following specific steps: firstly, absorbing and removing supernatant, adding PBS (0.01M, pH7.4) to clean cells for 3 times, then adding 600 mul of precooled paraformaldehyde with volume fraction of 4% into each hole, and fixing the cells for 30min at normal temperature; after fixation, removing paraformaldehyde by pipetting, washing cells with PBS for 3 times, washing, incubating, adding 600 μ l of His-tag (4℃ 2) monoclonal antibody (purchased from Biowort ECHNOLOGY) diluted with PBS at a volume ratio of 1:5000, and standing in a refrigerator at 4 deg.C overnight; after the primary antibody incubation is finished, washing cells for 3 times by using PBS, adding 600 mu l of FITC labeled goat anti-mouse secondary antibody (purchased from Dingguo biological Co., Ltd.) diluted by using PBS according to the volume ratio of 1:100 for incubation, and reacting for 1h in a dark place at 37 ℃; second antibody hatchingAfter incubation, cells were washed 3 times with PBS, 1mL PBS was bottom-sealed, and observed under an inverted fluorescence microscope, with the results shown in fig. 4: FIG. 4a shows the result of negative control group, and FIG. 4b shows the result of (P1) BV-Flic infecting Sf9 cells. The results show that: the negative control group showed no fluorescence, and the specific green fluorescence was evident in BV-Flic infected Sf9 cells. It is shown that the BV-Flic infected Sf9 cells can successfully express Flic-B protein.

1.5 purification and characterization of Flic-B protein

(1) Obtaining and condition optimization of Flic-B protein

The cell density of P2 generation recombinant baculovirus infected with

MOI

1, 3 and 5 was 2.5 × 10⁶Carrying out suspension culture on sf9 cells per ml at the table temperature of 27 ℃ and the rotating speed of 100-120 rpm for 72h, collecting the cells, and carrying out ultrasonic treatment under the following treatment conditions: working time is 3s, intermittent time is 5s, working power is 200w, and crushing time is 10 min. The cell suspension after ultrasonic treatment becomes clear and transparent, the treated cell suspension is centrifuged at 10000rpm for 8-10 min at 4 ℃ to obtain supernatant, the treated Flic-B protein exists in the supernatant in the form of soluble protein, a proper amount of protein sample is taken for sample preparation, SDS-PAGE and Western-Blot identification are carried out, the result is shown in figures 5 and 6, an obvious protein band is shown at 53.6KDa, the protein band is consistent with the expectation, the protein expression is optimal when MOI is 5, and the protein is stored for subsequent purification.

The SDS-PAGE was specifically performed as follows:

firstly, preparing 12% of protein gel according to an SDS-PAGE gel kit (purchased from Dingguo biology Co., Ltd.); adding the protein sample into 6 Xprotein loading buffer solution in proportion, boiling in boiling water for 10min, and centrifuging instantly for later use; placing the protein gel in an electrophoresis tank, sequentially adding a protein sample and a protein Maker, inserting electrodes, performing 80V isobaric electrophoresis for about 30min after electrifying, adjusting the voltage to 120V isobaric electrophoresis for about 90min, and allowing bromophenol blue in SDS loading buffer solution to run to the lowest position of the gel; dyeing and decoloring: after electrophoresis, putting the albumin glue into a Coomassie brilliant blue solution for dyeing for 30min, and adding a decolorizing solution for treatment after dyeing; and (4) observation: and (3) placing the completely decolorized albumin glue in a two-color laser imaging split system for scanning.

Western-Blot was performed as follows:

firstly, polyacrylamide gel electrophoresis is carried out according to the method, and after the electrophoresis is finished, Western-Blot is carried out according to the following steps: cutting the glue: cutting off corresponding colloid according to the size of target protein by referring to a protein Marker, preparing an NC membrane with similar size, putting the NC membrane into a mold transferring solution, and soaking for 10 min; preparing a film transfer: preparing a film conversion instrument, sequentially putting a black surface, a spongy cushion, three layers of filter paper, gel, an NC film, three layers of filter paper, a spongy cushion and a white surface in sequence, clamping, putting into the electrotransformation instrument, inserting an electrode, and placing ice blocks around the electrotransformation instrument for cooling; film transfer: setting the electrophoresis apparatus at a constant current of 200mA, and electrically switching for 70 min; and (3) sealing: after the electrotransformation is finished, taking out the NC membrane, placing the NC membrane in a 5% skimmed milk powder solution, and sealing the NC membrane on a shaking table at room temperature for 2 hours; washing the membrane: after the sealing is finished, removing the sealing liquid, adding PBST (poly-p-phenylene benzobisoxazole) for cleaning for 10min, removing the PBST solution, and repeating the operation for three times; incubating the primary antibody: after the above operation is completed, adding His-tag (4℃ 2) monoclonal antibody (purchased from BiowordECHNOLOGY) diluted by PBS according to the volume ratio of 1:5000, fully soaking the membrane, placing the membrane in a refrigerator at 4 ℃ for incubation overnight, and washing the membrane; incubation of secondary antibody: after the previous operation is finished, adding TBS in a dark place according to a volume ratio of 1: 10000 diluted

800CW Goat anti-Mouse IgG (H + L) second Antibody (from LI-COR Biosciences), shaking-incubated at room temperature for 1H, and washing; and (4) observing results: and (4) placing the processed NC film into a two-color laser imaging split system for scanning and photographing, storing pictures and analyzing results.

(2) Purification and characterization of Flic-B protein

Purifying the prepared protein solution by adopting a Ni-NAT affinity chromatography, and specifically operating as follows:

firstly, the hollow column containing Ni-NTA is treated with ddH₂O, adding 1ml of Ni-NTA to the column after rinsing, and adding ddH₂Cleaning O, and dripping by gravity;

sealing the lower end of the column, adding the prepared Flic-B protein solution into the column, reversing the upper part and the lower part to fully combine the nickel and the protein solution, placing at 4 ℃ for 30min, uniformly mixing every 5-6min, taking 100 μ l of stock solution preparation sample for SDS-PAGE identification, and obtaining the result shown in lane 1 of FIG. 7;

after the protein solution is fully combined with the nickel, the solution dropped under the action of gravity is the unassociated part of the protein solution, and 100 mul of the solution preparation sample is taken for SDS-PAGE identification, as shown in a lane 2 of a figure 7;

the target protein was eluted with 20mM, 50mM, 100mM, 200mM, 300mM, 400mM, and 500mM of imidazole, and 100. mu.l of each eluate was sampled for SDS-PAGE, as shown in lanes 3-9 of FIG. 7, wherein lane M is a 170kDa protein Maker.

The results show that: the SDS-PAGE identification shows that a distinct protein band is shown at 53.6KDa, and is consistent with the expectation. At the same time, the results show that a large amount of hetero-protein and a small amount of target protein can be eluted by using 100mM imidazole, and a large amount of single target protein can be eluted by using 500mM imidazole. Thus, the purification results for adjuvant preparation are shown in FIG. 8, wherein lane M is 170kDa protein Maker, lane 1 is crude protein liquid Flic-B, lane 2 is not column-like protein, lane 3 is 100mM imidazole-eluted hetero-protein, lanes 4-5 are 200mM imidazole-eluted single band protein Flic-B, lane 6 is 500mM imidazole-eluted single band protein Flic-B, and the results show that 200mM imidazole and 500mM imidazole both elute a single band protein of interest after 100mM imidazole-eluted hetero-protein.

EXAMPLE 2 construction and expression of recombinant plasmid of Salmonella typhimurium flagellin Flic-E based on E.coli expression System

2.1 Synthesis and amplification of Salmonella typhimurium flagellin Flic Gene

This procedure is the same as in example 1.1.

2.2 construction and identification of pET-32a-Flic recombinant expression vector

And (3) recovering the PCR product, performing double enzyme digestion on the gel by using BamH I and Hind III to recover a target product and a pET-32a empty vector at 37 ℃ for 5h, connecting the target gene and the vector at 16 ℃ overnight after gel recovery, and transforming the connecting product into escherichia coli DH5a competent cells to obtain the vector pET-32 a-Flic. The recombinant plasmid is identified by a double enzyme digestion mode, and the plasmid is sequenced, so that a correct result is obtained, and the recombinant expression vector pET-32a-Flic is obtained, as shown in figure 9, a Lane M is DNAmaker with the size of 5000, and a Lane 1 is an enzyme digestion identification result.

2.3 construction of recombinant expression bacterium pET-32a-Flic/BL21

The recombinant plasmid pET-32a-Flic is transformed into BL21(DE3) pLysS competent cells to obtain a positive clone strain, namely a recombinant expression strain named pET-32a-Flic/BL 21.

2.4 obtaining and identifying recombinant protein Flic-E

The recombinant expression strain pET-32a-Flic/BL21 was inoculated into LB liquid medium containing 100. mu.g/mL Amp + (ampicillin) at a volume ratio of 1: 1000, and cultured overnight at 37 ℃ with shaking at 220rpm, to give a seed solution. Expanding the seed liquid during induction expression, transferring the cultured seed liquid into Amp + fresh LB liquid culture medium according to the volume ratio of 1:100, culturing the seed liquid on a shaking table at 37 ℃ and 220rpm for about 3-4 h to ensure that the OD of the bacterial liquid is₆₀₀The nm value is about 0.6, and an inducer IPTG can be added for induction, wherein the induction conditions are as follows: IPTG concentration was 0.1mM, temperature was 30 ℃, rotation speed was 150rpm, and induction time was 12 h. As shown in fig. 10 and 11, the bacterial solution with induced expression was subjected to ultrasonic treatment under the following conditions: working time is 3s, intermittent time is 5s, working power is 200w, and crushing time is 10 min. And (3) clarifying and brightening the bacteria liquid after ultrasonic treatment, centrifuging the treated bacteria liquid at 4 ℃ and 10000rpm for 8-10 min, taking supernatant, allowing the treated Flic-E protein to exist in the supernatant in a soluble protein form, taking a proper amount of protein Flic-E to prepare a sample, and performing SDS and Western-Blot identification. The induction temperatures were set at 27 ℃, 30 ℃ and 37 ℃ for induction expression, and as a result, as shown in FIG. 12, it was found that the target protein showed a target band at 60 to 75kDa due to the difference in expression system, and the expression level was the highest when the temperature was set at 30 ℃.

2.5 purification and characterization of Flic-E protein

The purification procedure was the same as that of example 1.5(2), and the results of SDS assay are shown in FIG. 13, which shows that lane 3 eluted with 100mM imidazole as the hetero protein and that a large amount of the hetero protein was eluted under these conditions, that

lanes

4 and 5 eluted with 200mM imidazole as the Flic-E protein, although a large amount of the hetero protein was eluted, contained a portion of the hetero protein and the purification effect was inferior to that of Flic-B expressed by the baculovirus expression system, and that lane 6 eluted with 500mM imidazole as the sample band, and that no band was found, indicating that the Flic-E protein expressed by the E.coli expression system was eluted completely at 200mM imidazole.

EXAMPLE 3BCA assay for determining Flic-B and Flic-E protein concentrations

Protein concentrations of Flic-B and Flic-E were determined using the BCA kit (Thermo Scientific) as follows:

(1) preparing a working solution: according to the standard and the sample quantity, according to the reagent A: reagent B at a volume ratio of 50: 1, preparing a proper amount of BCA working solution, and fully and uniformly mixing, wherein the working solution is used as the working solution, and 200 mu L of the working solution is needed by each hole;

(2) diluting the standard substance: bovine Serum Albumin (BSA) standard at 2mg/mL (A) stock was diluted with imidazole solution of the same concentration as the eluted protein solution according to the following table:

(3) respectively adding 25 mu L of standard substance and purified Flic-B and Flic-E protein samples into a 96-well plate correspondingly, and repeating 3 times for each sample;

(4) adding 200 mu L of prepared BCA working solution into each hole, and fully and uniformly mixing;

(5) covering a 96-well plate cover, and incubating at 37 ℃ for 30 min;

(6) and (5) cooling to room temperature, measuring the wavelength of 562nm by using a microplate reader, and calculating the concentrations of purified Flic-B and Flic-E proteins according to a standard curve.

And (4) analyzing results: according to the calculation of a standard curve, 20ml of cells can obtain about 11.28mg of Flic-B protein after expression and purification; after 20ml of bacterial liquid is induced, expressed and purified, about 3mgFlic-E protein can be obtained; as a result, it was found that: the expression level of Flic-B protein expressed by the baculovirus expression system is much higher than that of Flic-E protein expressed by the Escherichia coli expression system.

Example 4 immunogenicity assay

4.1 preparation of H9N2 inactivated avian influenza vaccine containing Salmonella typhimurium flagellin Flic-B and Flic-E adjuvants

Flic with a single protein fluid of interest purified in examples 1 and 2, respectively, was completely inactivated with a final concentration of 0.2% formalin for H9N2 virus (A/chicken/Guangdong/V/2008 (V)_K627) The virus strain is disclosed in the literature "MengYu et al. Expression pattern of NLRP3 and its related cytokines in the lung and library of influenza virus H9N2 infested BALB/c mica, which is disclosed in Virology Journal (2014)11: 229" in a volume ratio of 1:8 as an aqueous phase, and the aqueous phase of the vaccine is emulsified with SEPPICMINIDETM ISA201VG adjuvant in a volume ratio of 1:1 to obtain the H9N2 inactivated avian influenza vaccine with a Flic adjuvant content of 20. mu.g/0.3 ml. Wherein the content of H9N2 virus in each 0.1ml of inactivated vaccine is not less than 5 x 10 before inactivating⁷EID 50, the content of the inactivated vaccine is consistent with the virus content of the commercial H9N2 inactivated vaccine, and the commercial H9N2 inactivated vaccine is a white emulsion vaccine.

4.2 immunization procedure

52 SPF chickens (purchased from Xinghua agricultural fowl eggs, Inc., of Guangdong province) were randomly divided into 4 groups of 14 chickens, namely PBS group, H9N2 commercial inactivated vaccine group, H9N2 inactivated vaccine + Flic-B group, and H9N2 inactivated vaccine + Flic-E group. The immunization mode is neck subcutaneous immunization, and the immunization dose is 300 mu l/mouse. Immunizing each group of SPF chickens at the age of 2 weeks, collecting blood from jugular veins at 7d, 14d and 21d after immunization respectively, and detecting HI antibody titer in serum so as to evaluate the humoral immune response level of the immunized organism; peripheral blood lymphocytes were isolated 3 weeks after immunization and lymphocyte proliferation was measured for evaluation of the level of adjuvant-stimulated cellular immune response.

(1) Detection of humoral immune response levels

Serum HI antibody titer detection: and (3) taking a 96-hole V-shaped plate, adding 25 mu L PBS into the 1 st to 11 th holes, and adding 50 mu LPBS into the 12 th hole. Diluting 25 μ L serum to 10 th well in multiple serial ratios, discarding 25 μ L, adding 25 μ L serum containing 4 HA unitsThe H9N2 avian influenza antigen (11 th well as an antigen control) was mixed, left to stand at room temperature for 30 minutes, and 1% of chicken red blood cells (12 th well as an erythrocyte control) were added to all the wells in equal amounts, and after standing at room temperature for 30 minutes, the highest dilution of serum that could completely inhibit the agglutination of erythrocytes was the HI titer of the test serum. The HI antibody titer levels at different time points of each of the above immunization groups are shown in fig. 14. The figure shows that no HI antibody titer was produced in the PBS group at three weeks after immunization, and that the H9N2 commercial inactivated vaccine group produced antibodies starting at week 1 with an average HI antibody titer of about 2^0.67 Week 2 antibody titer reached 2^6.83The antibody titer reached the highest at week three, about 2^8.08. While the H9N2 inactivated vaccine + Flic-B group and the H9N2 inactivated vaccine + Flic-E group rapidly produced antibodies 1 week after immunization, and the HI antibody titers were about 2^4.91And 2^4.17Has obvious difference with commercial inactivated vaccine group, and the antibody titer generated in the third week after immunization is the highest and is about 2^11.25And 2^10.83The results show that H9N2 inactivated avian influenza vaccine containing salmonella typhimurium flagellin Flic adjuvant produced antibodies rapidly after immunization and produced earlier, higher HI antibody titers than the commercial vaccine. Thus, the use of Flic adjuvant can obviously enhance the immunogenicity of the antigen. However, the HI antibody titer of the H9N2 inactivated vaccine + Flic-B group is higher than that of the H9N2 inactivated vaccine + Flic-E group at different time points after immunization, which indicates that the Flic-B protein expressed by the baculovirus expression system has better adjuvant effect and higher biological activity.

(2) Detection of cellular immune response levels

MTT method for detecting the proliferation of peripheral blood lymphocytes: 3 chickens were collected from each group 3 weeks after immunization, 2mL of blood was collected from the jugular vein of the immunized chicken into an anticoagulation tube, and peripheral blood lymphocytes were separated according to the instructions. And determining the level of lymphocyte proliferation according to MTT cell activity assay instructions. With 3 replicates per sample. The proliferation results of the peripheral blood lymphocytes after 3 weeks of immunization in each immunization group are shown in fig. 15, and the results show that the proliferation level of the PBMC of the H9N2 inactivated vaccine + Flic-B group is the highest, and the proliferation level of the PBMC of the H9N2 inactivated vaccine + Flic-E group is the next, compared with that of the commercial vaccine group, so that the cellular immune response of the organism can be obviously enhanced under the action of Flic, and the cellular immunity generated by the organism is more obviously stimulated under the effect of Flic-B adjuvant.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> baculovirus expression system-based Flic immune adjuvant, preparation method and application

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>494

<212>PRT

<213> Salmonella typhimurium (Salmonella typhimurium)

<220>

<223> amino acid sequence of Salmonella typhimurium flagellin Flic

<400>1

Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn Asn

1 5 10 15

Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu Ser

20 25 30

Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala

35 40 45

Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser

50 55 60

Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala

65 70 75 80

Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala Val

85 90 95

Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile Gln

100 105 110

Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly Gln

115 120 125

Thr Gln Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu Thr

130 135 140

Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys

145 150 155 160

Gln Ile Asn Ser Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln

165 170 175

Lys Tyr Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp

180 185 190

Thr Thr Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly

195 200 205

Leu Gly Gly Thr Asp Gln Lys Ile Asp Gly Asp Leu Lys Phe Asp Asp

210 215 220

Thr Thr Gly Lys Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr Gly

225 230 235 240

Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp Lys Thr Asn Gly Glu Val

245 250 255

Thr Leu Ala Gly Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro Ala

260 265 270

Thr Ala Thr Glu Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp Leu

275 280 285

Thr Glu Ala Lys Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr Ala

290 295 300

Ser Val Val Lys Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile Asp

305 310 315 320

Gly Gly Leu Ala Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala Thr Gln

325 330 335

Asn Lys Asp Gly Ser Ile Ser Ile Asn Thr Thr Lys Tyr Thr Ala Asp

340 345 350

Asp Gly Thr Ser Lys Thr Ala Leu Asn Lys Leu Gly Gly Ala Asp Gly

355 360 365

Lys Thr Glu Val Val Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser Lys

370 375 380

Ala Glu Gly His Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala Ala

385 390 395 400

Ala Thr Thr Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala

405 410 415

Gln Val Asp Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe

420 425 430

Asn Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser

435 440 445

Ala Arg Ser Arg Ile Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn

450 455 460

Met Ser Arg Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala

465 470 475 480

Gln Ala Asn Gln Val Pro Gln Asn Val Leu Ser Leu Leu Arg

485 490

<210>2

<211>1488

<212>DNA

<213> Salmonella typhimurium (Salmonella typhimurium)

<220>

<223> encoding nucleotide sequence of Salmonella typhimurium flagellin Flic

<400>2

atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60

tcccagtccg ctctgggcac cgctatcgag cgtctgtctt ccggtctgcg tatcaacagc 120

gcgaaagacg atgcggcagg tcaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180

ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240

gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgct 300

aacagcacca actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360

aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420

gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tatcgatctg 480

aagcagatca actctcagac cctgggtctg gatacgctga atgtgcaaca aaaatataag 540

gtcagcgata cggctgcaac tgttacagga tatgccgata ctacgattgc tttagacaat 600

agtactttta aagcctcggc tactggtctt ggtggtactg accagaaaat tgatggcgat 660

ttaaaatttg atgatacgac tggaaaatat tacgccaaag ttaccgttac ggggggaact 720

ggtaaagatg gctattatga agtttccgtt gataagacga acggtgaggt gactcttgct 780

ggcggtgcga cttccccgct tacaggtgga ctacctgcga cagcaactga ggatgtgaaa 840

aatgtacaag ttgcaaatgc tgatttgaca gaggctaaag ccgcattgac agcagcaggt 900

gttaccggca cagcatctgt tgttaagatg tcttatactg ataataacgg taaaactatt 960

gatggtggtt tagcagttaa ggtaggcgat gattactatt ctgcaactca aaataaagat 1020

ggttccataa gtattaatac tacgaaatac actgcagatg acggtacatc caaaactgca 1080

ctaaacaaac tgggtggcgc agacggcaaa accgaagttg tttctattgg tggtaaaact 1140

tacgctgcaa gtaaagccga aggtcacaac tttaaagcac agcctgatct ggcggaagcg 1200

gctgctacaa ccaccgaaaa cccgctgcag aaaattgatg ctgctttggc acaggttgac 1260

acgttacgtt ctgacctggg tgcggtacag aaccgtttca actccgctat taccaacctg 1320

ggcaacaccg taaacaacct gacttctgcc cgtagccgta tcgaagattc cgactacgcg 1380

accgaagttt ccaacatgtc tcgcgcgcag attctgcagc aggccggtac ctccgttctg 1440

gcgcaggcga accaggttcc gcaaaacgtc ctctctttac tgcgttaa 1488

<210>3

<211>1482

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> optimized coding nucleotide sequence of salmonella typhimurium flagellin Flic

<400>3

gctcaggtca ttaacactaa ctccctgagc ctgctgactc agaacaacct gaacaagtcc 60

cagagcgctc tgggtaccgc tatcgaacgt ctgtccagcg gcctgcgcat caactccgct 120

aaggacgacg ctgctggcca ggccatcgcc aaccgcttca ccgctaacat caagggtctg 180

acccaggcct cccgcaacgc caacgacggt atctccatcg ctcagaccac cgaaggcgcc 240

ctgaacgaaa tcaacaacaa cctgcagcgc gtgcgcgagc tggccgtcca atccgctaac 300

tccactaaca gccagagcga cctggacagc atccaggctg agatcaccca gcgcctgaac 360

gagatcgacc gcgtgtccgg tcagacccag ttcaacggcg tcaaggtgct ggctcaggac 420

aacactctga ctatccaggt cggcgctaac gacggcgaga ccatcgacat cgacctgaag 480

cagatcaaca gccagaccct gggtctggac actctgaacg tgcagcagaa gtacaaggtg 540

agcgacactg ccgccaccgt cactggctac gctgacacca ccatcgccct ggacaactcc 600

actttcaagg cttccgccac tggcctgggt ggtactgacc agaagatcga cggtgacctg 660

aagttcgacg acaccaccgg caagtactac gccaaggtca ctgtgaccgg cggcactggc 720

aaggacggtt actacgaggt ctccgtggac aagactaacg gtgaagtgac cctggctggt 780

ggcgctacca gccccctgac tggtggcctg cctgccaccg ctactgaaga cgtgaagaac 840

gtccaggtgg ctaacgccga cctgaccgaa gccaaggctg ctctgactgc cgctggtgtg 900

actggtactg cttccgtggt caagatgtcc tacaccgaca acaacggcaa gaccatcgac 960

ggtggcctgg ctgtgaaggt gggtgacgac tactactccg ccacccagaa caaggacggt 1020

agcatctcca tcaacactac caagtacacc gccgacgacg gcaccagcaa gaccgctctg 1080

aacaagctgg gtggcgctga cggcaagacc gaagtggtct ccatcggtgg taaaacctac 1140

gctgctagca aggccgaggg tcacaacttc aaggctcagc ctgacctggc cgaagctgcc 1200

gccaccacca ccgagaaccc tctgcagaag atcgatgctg ctctggccca ggtggacact 1260

ctgcgcagcg acctgggtgc tgtccagaac cgtttcaaca gcgccatcac taacctgggt 1320

aacaccgtga acaacctgac tagcgctcgc agccgtatcg aggactccga ctacgccact 1380

gaggtgtcca acatgagccg cgctcagatc ctgcagcagg ccggtactag cgtgctggcc 1440

caggccaacc aggtccccca gaacgtcctg agcctgctcc gt 1482

<210>4

<211>1524

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding nucleotide sequence for optimized application of salmonella typhimurium flagellin Flic

<400>4

atgcatcacc atcaccatca cgctcaggtc attaacacta actccctgag cctgctgact 60

cagaacaacc tgaacaagtc ccagagcgct ctgggtaccg ctatcgaacg tctgtccagc 120

ggcctgcgca tcaactccgc taaggacgac gctgctggcc aggccatcgc caaccgcttc 180

accgctaaca tcaagggtct gacccaggcc tcccgcaacg ccaacgacgg tatctccatc 240

gctcagacca ccgaaggcgc cctgaacgaa atcaacaaca acctgcagcg cgtgcgcgag 300

ctggccgtcc aatccgctaa ctccactaac agccagagcg acctggacag catccaggct 360

gagatcaccc agcgcctgaa cgagatcgac cgcgtgtccg gtcagaccca gttcaacggc 420

gtcaaggtgc tggctcagga caacactctg actatccagg tcggcgctaa cgacggcgag 480

accatcgaca tcgacctgaa gcagatcaac agccagaccc tgggtctgga cactctgaac 540

gtgcagcaga agtacaaggt gagcgacact gccgccaccg tcactggcta cgctgacacc 600

accatcgccc tggacaactc cactttcaag gcttccgcca ctggcctggg tggtactgac 660

cagaagatcg acggtgacct gaagttcgac gacaccaccg gcaagtacta cgccaaggtc 720

actgtgaccg gcggcactgg caaggacggt tactacgagg tctccgtgga caagactaac 780

ggtgaagtga ccctggctgg tggcgctacc agccccctga ctggtggcct gcctgccacc 840

gctactgaag acgtgaagaa cgtccaggtg gctaacgccg acctgaccga agccaaggct 900

gctctgactg ccgctggtgt gactggtact gcttccgtgg tcaagatgtc ctacaccgac 960

aacaacggca agaccatcga cggtggcctg gctgtgaagg tgggtgacga ctactactcc 1020

gccacccaga acaaggacgg tagcatctcc atcaacacta ccaagtacac cgccgacgac 1080

ggcaccagca agaccgctct gaacaagctg ggtggcgctg acggcaagac cgaagtggtc 1140

tccatcggtg gtaaaacctacgctgctagc aaggccgagg gtcacaactt caaggctcag 1200

cctgacctgg ccgaagctgc cgccaccacc accgagaacc ctctgcagaa gatcgatgct 1260

gctctggccc aggtggacac tctgcgcagc gacctgggtg ctgtccagaa ccgtttcaac 1320

agcgccatca ctaacctggg taacaccgtg aacaacctga ctagcgctcg cagccgtatc 1380

gaggactccg actacgccac tgaggtgtcc aacatgagcc gcgctcagat cctgcagcag 1440

gccggtacta gcgtgctggc ccaggccaac caggtccccc agaacgtcct gagcctgctc 1500

cgtcatcacc atcaccatca ctaa 1524

<210>5

<211>54

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer P1

<400>5

cgggatccat gcatcaccat caccatcacg ctcaggtcat taacactaac tccc 54

<210>6

<211>52

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer P2

<400>6

cccaagcttt tagtgatggt gatggtgatg acggagcagg ctcaggacgt tc 52

Claims

1. A preparation method of Flic immune adjuvant based on baculovirus expression system is characterized by comprising the following steps: the soluble Flic protein is obtained by expressing and purifying a nucleotide sequence for coding salmonella typhimurium flagellin through a Bac-to-Bac baculovirus expression system.

2. The method for preparing Flic immunoadjuvant based on baculovirus expression system as claimed in claim 1, characterized by comprising the following steps:

(4) carrying out amplification culture on the first generation baculovirus;

3. The method for preparing Flic immunoadjuvant based on baculovirus expression system as claimed in claim 1 or 2, characterized in that: the amino acid sequence of the salmonella typhimurium flagellin comprises a sequence shown as SEQ ID NO. 1.

4. The method for preparing Flic immunoadjuvant based on baculovirus expression system as claimed in claim 1 or 2, characterized in that: the nucleotide sequence for coding the flagellin of the salmonella typhimurium comprises a sequence shown as SEQ ID NO. 3.

5. The method for preparing Flic immunoadjuvant based on baculovirus expression system as claimed in claim 2, characterized in that:

the transfer plasmid is pFastBac^TM1；

The Tn7 transposition recombination in the step (2) is realized by the following steps: transforming the recombinant transfer plasmid into a DH10Bac escherichia coli competent cell, transposing and recombining through Tn7, transposing and recombining a nucleotide sequence for coding salmonella typhimurium flagellin to a baculovirus shuttle vector Bacmid to obtain a recombinant baculovirus plasmid;

the transfection in the step (3) is a liposome transfection method;

the insect host cell in the step (3) is Sf9 cell;

the expanding culture in the step (4) means that the first generation of baculovirus is infected into insect host cells, the supernatant is the baculovirus, and the step of continuously infecting the obtained virus into the insect host cells is repeated according to actual requirements to obtain a larger amount of baculovirus;

the step (5) is as follows: infecting the second generation recombinant baculovirus with MOI of 1-5 to obtain 2.5 × 10 cell density⁶Carrying out suspension culture on sf9 cells per ml at 27-28 ℃ for 72h, and then collecting the cells;

6. The method for preparing Flic immunoadjuvant based on baculovirus expression system as claimed in claim 5, characterized in that:

the expanding culture in the step (4) comprises the following specific steps: the first generation of recombinant baculovirus was infected at a cell density of 2 × 10 at an MOI of 0.1⁶Carrying out suspension culture on sf9 cells per ml at 27-28 ℃ for 72h, and collecting cell culture supernatant, namely the second-generation recombinant baculovirus;

the conditions of the ultrasonic treatment are as follows: the working time is 3s, the intermittent time is 5s, the working power is 100-200 w, and the crushing time is 10-15 min;

the solid-liquid separation method is centrifugation;

the steps of the Ni-NAT affinity chromatography are as follows: and (3) putting the supernatant on a Ni-NAT column, fully mixing the supernatant and Ni uniformly, standing, eluting the hybrid protein by using 100mM imidazole after the unbound protein liquid flows out of the Ni-NAT column through the action of gravity, and directly eluting the target protein with His by using 200-500 mM imidazole to obtain the soluble Flic protein.

7. A Flic immunoadjuvant based on a baculovirus expression system, characterized in that: the preparation method of any one of claims 1 to 6.

8. Use of a Flic immunoadjuvant based on a baculovirus expression system as defined in claim 7 for the preparation of a vaccine.

9. Use according to claim 8, characterized in that it comprises the following steps: mixing the Flic immune adjuvant based on the baculovirus expression system with inactivated virus to serve as a vaccine water phase; and mixing the vaccine aqueous phase with a water-in-oil-in-water adjuvant to obtain the vaccine.

10. Use according to claim 9, characterized in that:

the virus is avian influenza virus; further H9N2 avian influenza virus;

the water-in-oil-in-water adjuvant is MONTANIDE ISA series adjuvant; further comprises MONTANIDETM ISA201VG adjuvant;

the vaccine comprises the following components: 50% volume of water-in-oil-in-water adjuvant, 20 mug/0.3 ml of Flic immune adjuvant based on baculovirus expression system, and not less than 5 x 10 of inactivated virus content before virus inactivation per 0.1ml⁷EID 50。