CN103169963A - Building and application of hydrophobia DNA (deoxyribonucleic acid) vaccine - Google Patents

Abstract

The invention provides a recombinant eukaryotic expression plasmid expressing rabies virus envelope glycoprotein (G protein) gene optimized according to mammalian codon preference as a rabies DNA vaccine.

Description

The construction and application of prv dna vaccine

Technical field

The present invention relates to the rabies vaccine field.More specifically, the present invention relates to the eukaryotic expression recombinant plasmid of RV membrane glycoprotein (G albumen) gene that a kind of expression as the prv dna vaccine optimizes by the inclined to one side preferendum of mammal codon.

Background technology

Rabies (Rabies) are by rabies virus (Rabies virus, RV) a kind of Zoonosis infectious disease that causes, popular in the world, take acute, gradual and lethal encephalitis as feature, in case the nervous symptoms case fatality rate occurs almost up to 100% ^[1,2]Every year is died from rabic number up to 5.5 ten thousand in the whole world, mainly occurs in the developing country in Asia and Africa ^[3,4]Since two thousand, China's Rabies presents again the trend of quick rise, and death toll was up to 3,300 people in 2007 ^[5], slightly descend subsequently, but still maintain higher level, caused 1,879 people dead in 2011 ^[6], become a serious public health problem.According to statistics, 99% above human world rabies death is because of due to the rabies lyssodexis ^[1]It is by vaccination that human world rabies are controlled effective measures, controls rabies from the source.Traditional rabies attenuated vaccine is with low cost, but has the potential safety hazard of the residual or pathogenic back mutation of virulence; Inactivated vaccine safety, effective, but cost is expensive ^[7,8]Therefore, development safety, effectively, animal rabies vaccine with low cost is still had a realistic meaning.

After the DNA vaccination immune animal, can induce simultaneously humoral immunization and cellular immunization, the attack of the opposing pathogen that can watch for animals.Simultaneously, DNA vaccination is easy to build, and a large amount of preparation technologies are simple, and low production cost is convenient to preserve, and need not refrigeration transportation, thereby causes the extensive concern of research worker ^[9,10]

Rabies virus (Rabies virus, RV) is Rhabdoviridae (Rhabdoviridae) Lyssavirus (Lssavirus) member ^[11]Rabies virus envelope protein (RVG) is receptor binding protein and one of the Major Virulence Factors of virus, is determining path and the pathogenicity of Virus entry nervous system or tissue, is also the major antigen of inducing the body protective immunological reaction ^[12]This research and establishment express the eukaryotic expression recombinant plasmid pCAGG-RVG of the codon optimized RVG gene of mammal, and its immunogenicity the target animals dog is assessed.

Summary of the invention

The present invention has built the eukaryotic expression recombinant plasmid pCAGG-RVG that expresses RVG albumen (RVG) gene of optimizing by the inclined to one side preferendum of mammal codon.Indirect immunofluorescence assay and Western blot result show, restructuring RVG obtains correction at pCAGG-RVG transfection bhk cell.With pCAGG-RVG by 500 μ g dosage through intramuscular injection path immunity beasle dog, 4 weeks of interval with same dose, approach booster immunization once and are detected serum RV neutralizing antibody in the forward and backward different time of initial immunity.As a result, the pCAGG-RVG primary immune response can be induced significant neutralizing antibody reaction, and the secondary immunity NAT shows reinforced effects significantly.After secondary immunity, NAT is through of short duration decline, i.e. it is steady that maintenance continues; the 54th week to the initial immunity; 7 immune dog NAT average out to 2.88IU, and all higher than 0.5IU, namely all higher than the minimum NAT requirement to the strong virus attack protection.To sum up, pCAGG-RVG has antirabic potentiality, is a kind of rabies candidate vaccine likely.

Particularly, the invention provides the following:

1. prv dna vaccine, described prv dna vaccine comprise the eukaryotic expression recombinant plasmid of expressing the rabies virus envelope glycoprotein gene of optimizing by the inclined to one side preferendum of mammal codon.

2. according to the 1st described prv dna vaccine, wherein said rabies virus envelope glycoprotein gene by the inclined to one side preferendum optimization of mammal codon has the sequence as shown in SEG ID No.1.

3. according to the 1st described prv dna vaccine, wherein said expression has sequence as shown in SEG ID No.2 by the eukaryotic expression recombinant plasmid of the rabies virus envelope glycoprotein gene of the inclined to one side preferendum optimization of mammal codon.

4. has the eukaryotic expression recombinant plasmid of the sequence as shown in SEG ID No.2 as the purposes of prv dna vaccine.

5. the purposes of eukaryotic expression recombinant plasmid in preparation prv dna vaccine that has the sequence as shown in SEG ID No.2.

Description of drawings

Fig. 1: the expression of indirect immunofluorescene assay RVG albumen in pCAGG-RVG transfection bhk cell.The bhk cell of A:pCAGG-RVG transfection; The bhk cell that B:RV attenuated vaccine strain ERA infects; The bhk cell of C:pCAGGS transfection.

Fig. 2: Western blotting detects the expression of RVG albumen in pCAGG-RVG transfection bhk cell.Being laid on 6 orifice plate density with the pCAGG-RVG of 2 μ g or pCAGGS transfection and being about 80% bhk cell, is 1 to infect transfection and be laid on 6 orifice plate density and be about 80% bhk cell with RV attenuated vaccine strain ERA by MOI simultaneously.After 36 hours, results, cell lysis carry out the SDS-PAGE electrophoresis with the cell lysis supernatant, after the protein transfer printing is to the nitrocellulose filter, carries out Western blot take mouse-anti RV serum as primary antibodie and analyze.M: protein molecular weight standard; The bhk cell of A:pCAGGS transfection; The bhk cell that B:RV attenuated vaccine strain ERA infects; The bhk cell of C:pCAGG-RVG transfection.

Fig. 3: the immune efficacy of prv dna vaccine pCAGG-RVG to dog.Press 500 μ g/1mL/ dosage only through 7 beasle dogs of intramuscular injection path immunity with the eukaryotic expression recombinant plasmid pCAGG-RVG that expresses RVG albumen, 4 weeks of interval are carried out booster immunization with same dose, approach.Before immunity, with the rear different time points acquisition test canine peripheral blood of immunity, separation of serum detects the special NAT of RV in serum by neutralization test.

The specific embodiment

The invention will be further described below in conjunction with specific embodiment.

Materials and methods

1. plasmid, serum, cell strain and strain

Carrier for expression of eukaryon pCAGGS is provided by doctor Y.Kawaoka ^[13](clearly disclosing the construction method of carrier pCAGGS in document 13); The anti-rabies virus standard serum is available from OIE rabies reference laboratory (France); (hamster nephrocyte) BHK-21:ATCC NO.CCL10; RV attenuated vaccine strain ERA:CVCC NO.AV61*; (RV attenuated vaccine strain ERA is by 10 for mouse-anti RV serum ⁶FFU/mL/ dosage immunity mice in 6 age in week only, 3 weeks of interval, booster immunization 2 times, after the 3rd immunity, 2 weeks gathered mouse blood, separation of serum); The source of the RV standard CVS11 of the fixed virus document that sees reference ^[14]

2. main agents and laboratory animal

Restricted enzyme, T4 ligase are all available from precious biological engineering (Dalian) company limited; Plasmid Giga Kits is available from QIAGEN company; Liposome Lipofectamine ^TM2000 transfection reagents are available from Invitrogen company; The anti-mouse IgG of rabbit of the anti-mouse IgG of rabbit of green fluorescein (FITC) labelling, horseradish peroxidase (HRP) labelling is all available from Sigma company.Beasle dog is available from Guangzhou medical industry research total institute's Animal Experimental Study development centre (state's domesticated dog class laboratory animal kind subcenter).

The structure of embodiment 1, eukaryon expression plasmid pCAGG-RVG and preparation

(its complete genome group sequence is seen No. GenBank: the FJ913470) gene order of membrane glycoprotein (G albumen) (original series before optimizing is seen SEG ID No.3) to optimize rabies virus attenuated vaccine strain ERA by the inclined to one side preferendum of mammal codon.One seed amino acid all has several codons, just says that also these codons all can translate into a seed amino acid.Mammal has inclined to one side preferendum to codon, it is low that the height that namely a kind of translation efficiency of amino acid whose several codons has in mammalian body has, in this article " optimization " be about to amino acid whose codon and all be rewritten as the high codon of translation efficiency in mammalian body.Original series is that with the difference of optimizing rear sequence the aminoacid sequence of translating into is constant, but nucleotide sequence changes especially greatly.Sequence after optimization is seen SEG ID No.1, this gene of synthetic, and introduce Kozak sequence (GCCGCCACC) before start codon, two ends are introduced EcoRI and XhoI restriction enzyme enzyme sequence (GAATTC and CTCGAG), be cloned into pUC57 carrier (GenScript NO.SD1176), obtain plasmid pUC57-RVG.With BamH I single endonuclease digestion pCAGGS, remove wherein SV40 ori sequence (SV40 replication initiation sequence), connect and compose plasmid pCAGGS Δ SV40 ori; Again with EcoRI and XhoI restriction enzymes double zyme cutting pUC57-RVG, obtain the RVG gene, and sub-clone is to the corresponding restriction enzyme site of pCAGGS Δ SV40 ori, the RVG protein gene is positioned at chicken β-actin transcripting promoter downstream, obtains to express the eukaryon expression plasmid pCAGG-RVG (SEG ID No.2) of RVG protein gene.Identify with EcoRI and XhoI single endonuclease digestion respectively, prepare in a large number plasmid pCAGG-RVG (concrete operations see the test kit description for details) by plasmid Giga Kits, be used for transfection and immunity test.

EcoRI and XhoI double digestion pCAGG-RVG obtain two fragments that size is about 1650bp and 4400bp, conform to expected results, show that the RVG protein gene obtains restructuring (result does not present) in eukaryon expression plasmid pCAGGS.

The transient transfection of embodiment 2, eukaryon expression plasmid pCAGG-RVG and indirect immunofluorescence (IFA) detect

In order to confirm that pCAGG-RVG expresses the antigenicity of RVG albumen, with pCAGG-RVG transfection bhk cell, before transfection, preparation 24 orifice plate grow overnight, density are about 80% bhk cell, and (concrete operations see Lipofectamine for details by the liposome transfection method ^TM2000 description) the pCAGG-RVG transfection of transfection 0.5 μ g to above-mentioned cell, turn then 5%CO ₂37 ℃ of cultivations.Simultaneously, the dosage as 0.1 infects bhk cell by MOI take RV attenuated vaccine strain ERA, as positive control; With the pCAGGS transfection bhk cell of 0.5 μ g, as negative control.After transfection 36 hours, wash cell 3 times with PBS, with 3% paraformaldehyde fixed cell 30min of pre-cooling; After PBS washed 3 times, 1%BSA sealed 20min; Adding the mouse-anti RV serum of 1: 100 times of dilution after PBST (0.05%Tween20) washing is primary antibodie, incubated at room 30min; The rabbit anti-mouse igg two anti-(Sigma NO.F9137) that adds 1: 200 times of dilution FITC labelling after the PBST washing, incubated at room 30min; After the PBS washing, be placed under fluorescence microscope (Leica DMIRES2) and observe.

Result shows, anti-RV Mus serum presents green positive fluorescence signal (Figure 1A) when detecting the bhk cell of pCAGG-RVG transfection, detect when bhk cell is infected in RV ERA strain and also present green positive fluorescence signal (Figure 1B), present negative fluorescence signal (Fig. 1 C) when detecting the bhk cell of pCAGGS transfection.Result shows, pCAGG-RVG expresses RVG albumen and has good reactionogenicity.

Embodiment 3, Western blotting (Western blot) are analyzed

Can express RVG albumen in order to analyze eukaryotic expression recombinant plasmid, be laid on the monolayer bhk cell of 6 orifice plates with the pCAGG-RVG transfection of 2 μ g.After transfection 36 hours, results, cell lysis added isopyknic 2 * SDS lysis buffer, boil 10min, after the centrifugal 10min of 10,000 * g, get supernatant and carry out SDS-PAGE (Bio-Rad) electrophoresis; With albumen electricity transfer printing (Bio-Rad) to nylon membrane (Ameresco), 5% skimmed milk sealing is spent the night, adding the mouse-anti RV serum of 1: 100 times of dilution after the PBST washing is primary antibodie, room temperature effect 1h, after the PBST washing, add 1: 2500 times of PBST dilution horseradish peroxidase (HR) labelling rabbit anti-mouse igg two anti-(Sigma NO.A9044) room temperature effect 1h, after the PBST washing, develop the color with DAB.

Western blotting demonstrates special 67ku and detects albumen, conforms to G albumen theoretical expected value (Fig. 2).Result shows, RVG albumen obtains to express in the bhk cell of pCAGG-RVG transfection.

Embodiment 4, serum neutralization test detect the DNA immunization dog and induce neutralizing antibody

In order further to estimate the immune efficacy of eukaryon expression plasmid pCAGG-RVG, (how all to inject same amount regardless of body weight with eukaryotic expression recombinant plasmid pCAGG-RVG by 500 μ g/1mL/ bars, PBS dilutes with phosphate buffer) dosage through negative beasle dog (12 monthly ages of beasle dog used of 7 RV neutralizing antibodies of intramuscular injection path immunity, dosage of inoculation and body weight are irrelevant, the injection site is the hind leg quadriceps femoris), 4 weeks of interval are again with same dose, approach booster immunization once.Simultaneously, with the identical negative beasle dog of 5 RV neutralizing antibodies of method immunity, 4 weeks of interval again with same dose, approach booster immunization once take pCAGGS as contrast.Gather blood, separation of serum respectively at the forward and backward different time points of initial immunity through the forelimb vein, be used for neutralizing antibody and detect.

The basic reference literature of the detection of RV neutralizing antibody ^[14]Carry out on 96 orifice plates.At first the blood serum sample that gathers is placed in 56 ℃ of water-bath 30min and carries out deactivation, more respectively with 9 times of dilutions of incomplete DMEM (Gibson), rear continuous 3 times of dilutions, every dilution factor volume is 50 μ L, approximately contains 100TCID with 50 μ L ₅₀The standard CVS11 of fixed virus virus liquid mix, after 1h was made in 37 ℃ of senses, every hole added approximately 10 ⁵Individual bhk cell is established the positive, feminine gender and cell contrast (positive control is standard serum, and initial serum dilution titer is 0.5IU/mL) simultaneously, and each dilution factor of serum is 4 parallel holes, 5%CO ₂Carry out IFA after 37 ℃ of cultivation 48h and detect, be placed on the fluorescence microscopy Microscopic observation, and calculate serum RV NAT.

In 4 weeks after initial immunity, just the RV neutralizing antibody can be detected in pCAGG-RVG immunity dog serum, and can't detect the RV neutralizing antibody in pCAGGS immunity dog serum.In 4 weeks after initial immunity, in pCAGG-RVG immunity dog serum, RV neutralizing antibody average titer rises to 3.23IU/mL (7.9,0.38,1.14,5.92,2.6,0.29 in 2 weeks after booster immunization, in immune dog serum, RV neutralizing antibody average titer rises to peak value 8.21IU/mL (13.50 and 4.5),, 7.79,13.5,4.5,5.92,7.79 RV neutralizing antibody level descends gradually subsequently and 4.5).In 27 weeks after initial immunity, in immune dog serum, RV neutralizing antibody average titer drops to 3.99IU/mL (7.79,3.24,2.6,2.6,2.6,0.87 and 1.14), yet in the 54th week after initial immunity, in immune dog serum, RV neutralizing antibody average titer still can reach 2.88IU/mL (7.79,2.6,2.6,2.6,2.6,0.87 and 1.14).These results show, pCAGG-RVG is a rabies candidate DNA vaccination (Fig. 3) with good immune efficacy.

Discuss

This research and establishment express the eukaryotic expression recombinant plasmid pCAGG-RVG of the codon optimized RV membrane glycoprotein G gene of mammal.Indirect immunofluorescence assay and Western blotting result show, RVG albumen obtains to express in the bhk cell of eukaryotic expression recombinant plasmid pCAGG-RVG transfection, and has good reactionogenicity.Use this eukaryotic expression recombinant plasmid as DNA vaccination immunity beasle dog, can induce remarkable and lasting RV neutralizing antibody reaction.

In immune serum, RV neutralizing antibody level is to estimate an important indicator of rabies vaccine immune efficacy.The results of study such as Osorio show, the DNA vaccination of expressing RV CVS strain G albumen with 1 dosage (100 μ g/ bar) only can be induced low-level RV neutralizing antibody reaction through intramuscular injection path immunity dog; Yet when immunizing dose is brought up to 300 μ g/ bar, can strengthen the reaction of RV neutralizing antibody ^[15]These result of study promptings, but the dependent RV neutralizing antibody reaction of prv dna vaccine immunity dog inductive dose; Increase immunizing dose and may obtain higher levels of RV neutralizing antibody reaction.Our result of study also confirms this supposition, with the dosage immunity dog of prv dna vaccine pCAGG-RVG by 500 μ g/, can induce significant RV neutralizing antibody, after initial immunity in 4 all immune dog serums RV NAT meansigma methods can reach 3.23IU/mL.

In addition, Perrin etc. study discovery: with the DNA vaccination of the expression RV PV strain G albumen of 1 dosage (100 μ g/ only) through intramuscular injection path immunity dog, only can induce low-level RV neutralizing antibody reaction, can strengthen the reaction of RV neutralizing antibody but the interval certain hour carries out booster immunization ^[16]This is consistent with this result of study, and we by 500 μ g/ dosage immunity dog only, can induce significant RV neutralizing antibody reaction with prv dna vaccine pCAGG-RVG, after initial immunity in 4 all immune dog serums RV NAT meansigma methods can reach 3.23IU/mL; 4 weeks of interval are carried out booster immunization with same dose, approach, can significantly strengthen RV neutralizing antibody reaction, after booster immunization in 2 all immune dog serums RV NAT meansigma methods up to 8.21IU/mL.

Although, the RV NAT peak averaging value (being respectively 70.4IU/mL, 96IU/mL and 96.71IU/mL) that this research is induced with the canine recombinant distemper virus rCDV-RVG (106TCID50/) of RVERA vaccine strain (106FFU/ is only), the recombinant Newcastle disease virus rLa-RVG (109.3EID50/ is only) that expresses RV G protein gene and expression RV G protein gene respectively in the research before us with RV NAT peak averaging value (8.21IU/mL) in prv dna vaccine pCAGG-RVG immunity dog serum ^[17,18]Yet in prv dna vaccine pCAGG-RVG immunity dog serum, RV NAT decline ground is more slow; after initial immunity in 54 all immune dog serums RV NAT meansigma methods still can reach 2.88IU/mL, and the RV NAT of 0.5IU/mL is can watch for animals to avoid the minimum titre that fatal dose RV street strain attacks ^[11,17,18]The immunity inoculation of this results suggest: pCAGG-RVG can provide effective protection more than 1 year for dog.Therefore, prv dna vaccine pCAGG-RVG is a rabies candidate vaccine likely.

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Claims (5)

1. prv dna vaccine, described prv dna vaccine comprise the eukaryotic expression recombinant plasmid of expressing the rabies virus envelope glycoprotein gene of optimizing by the inclined to one side preferendum of mammal codon.

2. prv dna vaccine according to claim 1, wherein said rabies virus envelope glycoprotein gene by the inclined to one side preferendum optimization of mammal codon has the sequence as shown in SEG ID No.1.

3. prv dna vaccine according to claim 1, wherein said expression has sequence as shown in SEG ID No.2 by the eukaryotic expression recombinant plasmid of the rabies virus envelope glycoprotein gene of the inclined to one side preferendum optimization of mammal codon.

4. has the eukaryotic expression recombinant plasmid of the sequence as shown in SEG ID No.2 as the purposes of prv dna vaccine.

5. the purposes of eukaryotic expression recombinant plasmid in preparation prv dna vaccine that has the sequence as shown in SEG ID No.2.

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