CN107344969A - A kind of nanometer influenza vaccines and construction method and application - Google Patents
A kind of nanometer influenza vaccines and construction method and application Download PDFInfo
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- CN107344969A CN107344969A CN201610292382.9A CN201610292382A CN107344969A CN 107344969 A CN107344969 A CN 107344969A CN 201610292382 A CN201610292382 A CN 201610292382A CN 107344969 A CN107344969 A CN 107344969A
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Abstract
The invention discloses a kind of nanometer influenza vaccines and construction method and application, described nanometer influenza vaccines are 3M2e rHF recombinant proteins, and its sequence is shown in SEQ ID NO.2.3M2e is illustrated in ferritin cage structure surface by the recombinant protein of the present invention, M2e immunogenicities is significantly improved, so as to construct a kind of new type influenza vaccine：3M2e rHF nano particles.The present invention utilizes prokaryotic expression system Bacillus coli expression recombinant protein vaccine, and vaccine preparation process is not related to live virus, more safe and simple compared to traditional chicken embryo preparation influenza vaccines method operation, is appropriate for quickly mass producing；Using M2e in different subtype influenza virus sequence it is well-conserved, it is experimentally confirmed that 3M2e rHF protection mouse have resisted the infection of homotype and special-shaped influenza virus, be expected to develop into the universal influenza vaccine with cross protection effect.
Description
Technical field
The present invention relates to vaccine manufacturing technology field, is more particularly to a kind of nanometer influenza vaccines, and described vaccine passes through in Ren Yuan
The tandem sequence repeats influenza m 2 e polypeptides of ferritin cage structure surface display three, M2e immunogenes can be significantly increased by preparing
Property, have cross protection effect nanometer influenza vaccines.
Background technology
Influenza virus is a kind of relatively common Respirovirus for endangering human health, and seasonal influenza is annual about in the whole world
Cause 300~5,000,000 people illness and 25~500,000 people dead.The appearance of potential new strains of influenza viruses and prevalence are good for the mankind
Kang Zaocheng is greatly threatened.
At present, vaccine inoculation is the maximally effective approach of flu-prevention.Influenza vaccines in use predominantly inactivate, attenuation or again
Group trivalent, tetravalence influenza vaccines, by induce produce for influenza virus major antigen HA or NA specific antibody come
Improve the ability of body resistance influenza infection.Because antigenic drift and difference persistently occur for HA, NA of influenza virus
Recombinated between influenza virus sub-strain, vaccine is required for updating every year.When seasonal influenza prevalence or influenza great outburst, it is difficult to
The sufficient vaccine of production in time.And the influenza virus in animal reservoir source, as avian influenza virus H 5 N 1, H7N9 and pig are flowed
Influenza Virus H1N1 across species propagation make exploitation have the universal influenza vaccine of broad spectrum protection effect extremely urgent.
Research to cross protection influenza vaccines is concentrated mainly on two conservative antigen epitopes of influenza A virus：Stromatin 2
Extracellular functional areas (M2e) and the peptidoglycan of hemagglutinin 2 (HA2gp).M2 is nonglycosylated homotetramer transmembrane protein,
Play proton channel in A types, Type B influenza virus.In influenza A, M2 albumen is made up of three fragments：Born of the same parents
Outer N- stub areas (M2e, 1~23 amino acid residue), transmembrane spanning α-helices and intracellular C- stub areas.With HA and N
A is compared, and M2e is highly conserved in different influenza As, thus as the potential target of Development of Universal influenza vaccines.
Nanometer technology prepares for vaccine and provides new strategy.A variety of nanometer materials, as polymer, liposome, nano-cluster,
Virus-like particle, ferritin are used to modified antigen to optimize its characteristic.Wherein, ferritin forms octahedron by 24 aggressiveness
Symmetrical albumen cage structure, it is widely present in life entity.People source ferritin is made up of two kinds of subunits：H chains (rHF, 21
) and L chains (19KDa) KDa.24 rHF subunits can also be self-assembly of albumen cage structure in vitro, and overall diameter is about
12nm, inside are the cavitys of 6-8 nanometers.The N-terminal of ferritin stretches to outer surface, is easy to carry out genetic modification, merges egg
Bletilla polypeptide.High-sequential repeats antigen and is easy to induce generation strong when microbial body surface is spaced apart with 5~10nm
The antibody response that T cell relies on.Distance is advantageous to open up in theory in the range of this between the neighboring amine groups end of ferritin surface
Show that antigen induction produces strong antibody response.Ferritin itself may also can play adjuvant effect, and human lymphocyte such as B is thin
Born of the same parents, CD4+T lymphocytes, CD8+T lymphocytic cell surfaces possess the specific binding sites of rHF, may be more beneficial for
Displaying antigen is identified by immunocyte.Moreover, ferritin has good resistance to heat, PH changes and chemical modification, have
There is high stability.Therefore, ferritin is the stable, nano-carrier of bio-compatible, is suitable for antigen displaying.The present invention
M2e polypeptides by the use of ferritin as carrier displaying multicopy, are expected to develop a kind of novel nano with cross protection effect
Influenza vaccines.
The content of the invention
The purpose of the present invention is to be to provide a kind of nanometer influenza vaccines, and described vaccine is 3M2e-rHF recombinant proteins, its egg
White matter sequence is shown in SEQ ID NO.2, and the gene order of coding 3M2e-rHF recombinant proteins is SEQ ID NO.1 institutes
Show.
It is another object of the present invention to provide the preparation method of a kind of nanometer of influenza vaccines, by people source ferritin H
The amino terminal of chain connects the M2e polypeptides (3M2e-rHF) of three sections of series connection, and restructuring egg is expressed using escherichia expression system
In vain, obtain showing the ferritin nano particle of 3M2e polypeptides after purification.
Final object of the present invention is the provision of application of a kind of nanometer of influenza vaccines in influenza virus vaccine is prepared.
In order to achieve the above object, the present invention takes following technical measures：
A kind of nanometer influenza vaccines, its protein sequence are shown in SEQ ID NO.2.
A kind of nanometer influenza vaccines, its preparation process include：
(1) sequence shown in SEQ ID NO.1, is inserted into plasmid pET28a, obtains recombinant plasmid pET28a/3M2e-rHF.
(2), recombinant plasmid pET28a/3M2e-rHF expression in escherichia coli and by molecular sieve column and sucrose density gradient centrifugation it is pure
Change albumen.
Application of a kind of nanometer of influenza vaccines in influenza virus vaccine is prepared, egg is recombinated using the 3M2e-rHF of the present invention
Combine in vain with other adjuvants and be prepared into influenza virus vaccine, or the 3M2e-rHF recombinant proteins of the present invention and other active ingredients
Combination is prepared as multiple vaccines.
The present invention compared with prior art, has advantages below and effect：
A, the present invention utilizes prokaryotic expression system Bacillus coli expression recombinant protein vaccine, and vaccine preparation process is not related to live virus,
It is more safe and simple compared to traditional chicken embryo preparation influenza vaccines method operation, it is appropriate for quickly mass producing；
B, adjuvant effect may be played by the use of people source ferritin as antigen display carrier, good biocompatibility, ferritin itself, shown
Write the immunogenicity for improving M2e antigens.The use of nano vaccine was prepared without additionally adding adjuvant so as to simplify vaccine
Journey；
C, vaccine of the present invention is immunized using collunarium approach, compared with conventional intramuscular injection immunization route, uses more secure side
Just, damage is small, and closer to the approach of influenza virus Natural invasive host, is advantageous to induction and produces cross protection effect；
D, the present invention using M2e in different subtype influenza virus sequence it is well-conserved, be expected to develop into and protect with intersecting
Protect the universal influenza vaccine of effect.
E, 3M2e-rHF nano particles.Under physiological condition, it is octahedra right that 3M2e-rHF nano particles are self-assembly of by 24 aggressiveness
Claim the hollow cage structure of system.Balb/c mouse are immunized through collunarium approach in 3M2e-rHF nano particles, and induction produces big
Measure the specific IgG antibodies of M2e.And 3M2e-rHF protection mouse have resisted the infection of homotype and special-shaped influenza virus.
Self assembly ferritin displaying conservative Antigenic Peptide provides new strategy for the exploitation of new generation vaccine.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of 3M2e-rHF nano particle vaccines；
Wherein a figures are 3M2e-rHF protein expression sequence construct schematic diagrames, and b is 3M2e-rHF nano particle assembling schematic diagrams.
Fig. 2 is the qualification figure of the 3M2e-rHF albumen of purifying.
A is the SDS-PAGE of the 3M2e-rHF albumen of purifying in Fig. 2.
B is the western blot qualification figures of the 3M2e-rHF albumen of purifying in Fig. 2.
Fig. 3 is the transmission electron microscope phenogram of 3M2e-rHF assemble nanometer particles.
Fig. 4 is that M2e specific IgG antibodies titre changes statistical chart in immune serum.
Fig. 5 a are Pneumovirinae titre statistical chart after A/Puerto Rico/8/34 (H1N1) influenza infection.
Fig. 5 b are mouse weight change statistical chart after A/Puerto Rico/8/34 (H1N1) influenza infection.
Fig. 5 c are mouse survival rate statistical chart after A/Puerto Rico/8/34 (H1N1) influenza infection.
Fig. 6 a are Pneumovirinae titre statistical chart after A/Chicken/Jiangsu/7/2002 (H9N2) influenza infection.
Fig. 6 b are mouse weight change statistical chart after A/Chicken/Jiangsu/7/2002 (H9N2) influenza infection.
Fig. 6 c are mouse survival rate statistical chart after A/Chicken/Jiangsu/7/2002 (H9N2) influenza infection.
Embodiment
The embodiment of the present invention be described in detail a kind of novel nano influenza vaccines preparation method and its immune effect in Mice Body
Verification process.Technical scheme described in the embodiment of the present invention, it is the conventional scheme of this area if not otherwise specified.
Embodiment 1：
The preparation method of 3M2e-rHF nanometer influenza vaccines, its step are：
(1) 3M2e-rHF protein expressing plasmids pET28a/3M2e-rHF is constructed：
PCR expands rHF sequences：With the plasmid pET-rHF of encoding human ferritin H chain-orderings (rHF) (by Dr Paolo
Santambrogio (Milan, Italy) give) it is template (Nanoscale, 2012,4,188), sense primer：5'
CCGGTGTAATGGAAGCTCCGACGGAGGAGGAGGATCTATGACGACCGCGTCCACCT
C 3', reverse primer：5'CCGCTCGAGTTAGCTTTCATTATCACTGTC 3'；
3M2e sequences：M2e nucleotide sequence (Gene ID are found in NCBI:956528), chemical synthesis 3M2e sequences.
Using the 3M2e nucleotide sequences of synthesis as template, sense primer：5'GGGAATTCCATATGATGTCTCTGCTG
ACCGAGG 3', reverse primer：5'GAGGTGGACGCGGTCGTCATAGATCCTCCTCCTCCG
TCGGAGCTTCCATTACACC 3', M2e amino acid sequence are MSLLTEVETPIRNEWGCRCNGSSD,
It is consistent with M2e sequences in A/Puerto Rico/8/34 (H1N1) influenza virus.
Using PCR primer rHF and 3M2e as template, Overlap-PCR amplifies 3M2e-rHF sequences, sense primer：
5'GGGAATTCCATATGATGTCTCTGCTGACCGAGG 3', reverse primer：5'CCGCTCGAG
TTAGCTTTCATTATCACTGTC 3', 98 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 1min 30s,
30 circulations of amplification, obtain the 3M2e-rHF nucleotide sequences shown in SEQ ID NO.1.Amplified production by Nde1 and
3M2e-rHF sequences are inserted into expression vector pET28a after Xho1 double digestions, construct 3M2e-rHF protein expression matter
Grain pET28a/3M2e-rHF.It is correct through sequence verification objective gene sequence.
(2) by plasmid pET28a/3M2e-rHF CaCl2Method is transformed into E. coli BL21 (λ DE3), uses LB
37 DEG C of culture medium, 180r/min cultivate thalline to OD600 between 0.4~0.6, add IPTG to final concentration of 1mM, 2
0 DEG C is continued to induce 16h, 4 DEG C, 6000g centrifugation 10min collection thalline, thalline is resuspended in into assembling buffer solution (20mM
Tris-HCl, 50mM NaCl, pH 8.0) in, after ultrasonication 40min, 10,000g centrifugation 30min, in collection
Clear to be put in water-bath 10min in 70 DEG C of water-baths, 10,000g centrifugation 30min remove precipitation.It is 1 to collect supernatant interception
After the concentration of 00KDa super filter tubes, collect destination protein through the 10/300GL of Superose 6 (being purchased from GE companies) molecular sieve column and wash
De- peak, protein concentrate pass through sucrose density gradient centrifugation, suction out destination protein layer, and 3M2e-rHF eggs are concentrated and purified out through dialysis
White nano particle.Bacterium solution of the 1L initial OD 600 0.4~0.6, through inducing after purification, it can about be purified into 0.5mg
3M2e-rHF albumen, the amino acid sequence of 3M2e-rHF albumen is shown in SEQ ID NO.2.
(3) purifying 3M2e-rHF albumen is characterized.Purifying protein is used for SDS-PAGE, with the rHF (21KD of purifying
A) compare (Fig. 2 a), it can be seen that 3M2e-rHF protein monomer bands between 35~40KDa.Use anti-ferriti
N heavy chain antibody (being purchased from Abcam Inc., Cambridge, MA) do primary antibody, and Western blot are further verified
3M2e-rHF (Fig. 2 b) expression.Transmission electron microscope (Fig. 3) is carried out to assembling 3M2e-rHF nano particles to characterize.Wherein
Transmission electron microscope sample preparation process is：Copper mesh absorption is adsorbed 2 on the drop containing 0.5mg/ml 3M2e-rHF nano particles
Min, nickel screen edge solution is gently sucked with filter paper, then dye 10min with 2% phosphotungstic acid.Copper mesh is placed natural in air
Dry, detected with 200kV transmission electron microscopes.
Embodiment 2：
Checking of the 3M2e-rHF nano particle influenza vaccines immune effects in Mice Body, its step are：
(1) 6~8 weeks big Balb/c mouse are grouped at random, respectively with 10 μ g 3M2e-rHF nano particles, 2.6 μ g
3M2e synthesis polypeptides (containing equimolar M2e compared with 3M2e-rHF nano particles), 6.3 μ g rHF
Nano particle (containing equimolar rHF compared with 3M2e-rHF nano particles) and PBS are right respectively through collunarium approach
Balb/c mouse are immunized, altogether it is immune three times, it is adjacent be immunized twice between be spaced three weeks.It is immune latter two weeks every time
Mice serum is gathered, is stored in -20 DEG C, ELISA detection M2e specific antibodies.Antibody test result shows three
Mouse is immunized as PBS control group mouse with rHF nano particles in secondary immune rear 3M2e polypeptides, in serum not
M2e specific IgG antibodies are detected, and the induction of 3M2e-rHF nano particles produces a large amount of M2e specific IgGs
Antibody (Fig. 4), it was demonstrated that the induction of 3M2e-rHF nano particles generates strong humoral immune response.
(2) mouse after being immunized one month three times, with 20 μ l 10LD50Homologous human influenza virus's strain A/Puerto Rico/8/3
4 (H1N1) and heterologous avian flu strain A/Chicken/Jiangsu/7/2002 (H9N2) difference infection immunity mouse,
Observation mouse weight change in continuous two weeks and survival rate, mouse weight reduce by more than 25% and are considered as death.Virus sense
The lung of 4 mouse of collection in the 4th day is contaminated, is detected for Pneumovirinae titre.
Pneumovirinae titer determination uses histocyte median infective dose (TCID50) method.Lung homogenate liquid 1000rpm centrifugations 10
Min, collect supernatant.2 × 10 are added into the every hole of 96 orifice plates4Individual mdck cell, cultivate in 37 DEG C of incubators
After 12 hours, infection mdck cell is cleaned with the lung homogenate liquid of 10 times of gradient dilutions, per the μ l of hole 100, often
The individual each concentration gradient of sample does 4 repetitions.After 37 ° of incubators are incubated 1h, the nothing of antibiotic is added with 200 μ l
Blood serum medium DMEM replaces cell culture supernatant, continues to be put into incubator culture three days, observes cell daily
Disease.After three days, the presence of influenza virus is determined with hemagglutination test (HA test), uses Reed＆Muench
Method is calculated virus titer.A/Puerto Rico/8/34 (H1N1) infection Pneumovirinae titer determination result is shown
The Pneumovirinae titre of 3M2e-rHF nano particle immune group mouse significantly reduces, and 3M2e polypeptides and rHF nanometers
The Pneumovirinae titre of grain immune group mouse does not have significant difference (Fig. 5 a) compared with PBS groups, illustrates 3M2e-rHF
Nano particle inhibits amplification of the virus in lung.After A/Puerto Rico/8/34 (H1N1) virus infection, 3M2e-
Just bottom out (Fig. 5 b), last survival rate are rHF nano particle immune group mouse weights after slight reduction
100% (Fig. 5 c), other immune group mouse are all dead in virus infection latter week, illustrate that 3M2e-rHF receives
Rice grain protection mouse has resisted the sexy dye of death of homotype influenza virus.A/Chicken/Jiangsu/7/2002(H9N2)
Infection Pneumovirinae titer determination result shows that the Pneumovirinae titre of 3M2e-rHF nano particle immune group mouse has dropped
It is low, although not significant difference (Fig. 6 a) compared with compareing immune group.After virus infection, 3M2e-rHF receives
Rice grain immune group mouse weight is infecting bottom out (Fig. 6 b) in the 8th day, and last survival rate is 100% (Fig. 6
C), and other immune group mouse weights are persistently reduced, it is all dead in virus infection latter week, illustrate 3M2e-r
HF nano particles protection mouse has resisted the sexy dye of death of special-shaped avian influenza virus.
SEQUENCE LISTING
<110>Wuhan Virology Institute,Chinan academy of Sciences
<120>A kind of nanometer influenza vaccines and construction method and application
<130>A kind of nanometer influenza vaccines and construction method and application
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 819
<212> DNA
<213>Artificial sequence
<400> 1
atgtctctgc tgaccgaggt ggaaacaccc atcaggaacg agtggggctg
cagatgtaat 60
gggagctccg acggatctgg aggaggagga atgagtctgc tgactgaggt
ggaaacccct 120
attcggaacg aatggggctg ccgctgtaat ggatctagtg atggaagcgg
aggaggagga 180
atgtccctgc tgacagaggt ggaaacccca attagaaacg agtggggctg
ccggtgtaat 240
ggaagctccg acggaggagg aggatctatg acgaccgcgt ccacctcgca
ggtgcgccag 300
aactaccacc aggactcaga ggccgccatc aaccgccaga tcaacctgga
gctctacgcc 360
tcctacgttt acctgtccat gtcttactac tttgaccgcg atgatgtggc
cttgaagaac 420
tttgccaaat actttcttca ccaatctcat gaggagaggg aacatgctga
gaaactgatg 480
aagctgcaga accaacgagg tggccgaatc ttccttcagg atatcaagaa
accagactgt 540
gatgactggg agagcgggct gaatgcgatg gagtgtgcat tacatttgga
aaaaaatgtg 600
aatcagtcac tactggaact gcacaaactg gccactgaca aaaatgaccc
ccatttgtgt 660
gacttcattg agacacatta cctgaatgag caggtgaaag ccatcaaaga
attgggtgac 720
cacgtgacca acttgcgcaa gatgggagcg cccgaatccg gcttggcgga
atatctcttt 780
gacaagcaca ccctgggaga cagtgataat
gaaagctaa
819
<210> 2
<211> 272
<212> PRT
<213>Artificial sequence
<400> 2
Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly
1
5
10
15
Cys Arg Cys Asn Gly Ser Ser Asp Gly Ser Gly Gly Gly Gly Met Ser
20
25
30
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg
35
40
45
Cys Asn Gly Ser Ser Asp Gly Ser Gly Gly Gly Gly Met Ser Leu Leu
50
55
60
Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn
65
70
75
80
Gly Ser Ser Asp Gly Gly Gly Gly Ser Met Thr Thr Ala Ser Thr Ser
85
90
95
Gln Val Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg
100
105
110
Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser
115
120
125
Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr
130
135
140
Phe Leu His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met
145
150
155
160
Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys
165
170
175
Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys
180
185
190
Ala Leu His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His
195
200
205
Lys Leu Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu
210
215
220
Thr His Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp
225
230
235 240
His Val Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala
245
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255
Glu Tyr Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser
260
265
270
Claims (5)
1. a kind of recombinant protein, its sequence is shown in SEQ ID NO.2.
2. encode the gene of albumen described in claim 1.
3. gene according to claim 2, its nucleotides sequence is classified as shown in SEQ ID NO.1.
4. the preparation method of recombinant protein described in claim 1, including：
（1）, by sequence shown in SEQ ID NO.1 insert plasmid pET28a, obtain recombinant plasmid pET28a/3M2e-rHF；
（2）, recombinant plasmid pET28a/3M2e-rHF expression in escherichia coli and purified by molecular sieve column and sucrose density gradient centrifugation, produce.
5. application of the recombinant protein in nanometer influenza vaccines are prepared described in claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111948589A (en) * | 2019-05-17 | 2020-11-17 | 南京林业大学 | Method for centrifugally separating ferritin nanoparticles by sucrose density gradient |
| CN112121161A (en) * | 2020-10-15 | 2020-12-25 | 北京理工大学 | Vaccine immunologic adjuvant, vaccine prepared by using vaccine immunologic adjuvant and preparation method of vaccine |
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