CN106011155A - Codon optimized severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn gene sequence carrying tPA signal peptide and nucleic acid vaccine thereof - Google Patents

Codon optimized severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn gene sequence carrying tPA signal peptide and nucleic acid vaccine thereof Download PDF

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CN106011155A
CN106011155A CN201610324251.4A CN201610324251A CN106011155A CN 106011155 A CN106011155 A CN 106011155A CN 201610324251 A CN201610324251 A CN 201610324251A CN 106011155 A CN106011155 A CN 106011155A
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sftsv
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李军
金柯
刘源
徐菱遥
韩亚萍
刘艳
周宜庆
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    • C12N2760/12011—Bunyaviridae
    • C12N2760/12211—Phlebovirus, e.g. Rift Valley fever virus
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Abstract

The invention belongs to the technical field of biological medicines, and relates to a codon optimized severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn gene sequence carrying tPA signal peptide and a nucleic acid vaccine thereof. The SFTSV glycoprotein Gn nucleic acid vaccine is composed of the codon optimized SFTSV glycoprotein Gn gene sequence and a eukaryotic expression vector pJW4303, and the 5'end of the SFTSV glycoprotein Gn gene sequence is connected with a tPA signal peptide sequence. Compared with a nucleic acid vaccine in a wild type state, the nucleic acid vaccine can express objective protein more efficiently and can effectively secrete the objective protein out of cells in gene expression, and an immune system can be effectively stimulated to produce good humoral immune response after a mammal is inoculated with the nucleic acid vaccine.

Description

Carry tPA signal peptide and codon optimized SFTSV Glycoprotein G n gene order and nucleic acid vaccine thereof
Technical field
The invention belongs to biomedicine technical field, relate to carrying tPA signal peptide and codon optimized serious heating companion's thrombocytopenia Syndrome virus aminoterminal Glycoprotein G n gene order and nucleic acid vaccine thereof.
Background technology
Serious heating companion's thrombocytopenic syndromes virus (severe fever with thrombocytopenia syndrome virus, SFTSV) it is Chinese scholar a kind of new virus of finding first in 2010 and identifying.Heating companion's thrombocytopenic syndromes (SFTS) It is to be infected a kind of emerging infectious disease with heating, thrombocytopenia, leukopenia as principal character caused, the state of an illness by SFTSV Severe patient may occur in which nervous system injury, phage-displayed peptide phenomenon, multiple organ dysfunction syndrome etc., and case fatality rate is up to 12%-16.3%, even Have been reported that to be 30%.
SFTSV belongs to bunyaviridae Phlebovirus.Genomic sequence analysis shows, its genome is sub-thread strand RNA, It is made up of large fragment L, middle fragment M and tri-fragments of small fragment S, is separately encoded the RNA polymerase (RdRp) that RNA relies on, Aminoterminal glycoprotein (Gn) and c-terminus glycoprotein (Gc), nucleoprotein (NP) and non-structural protein (NSs).New as one Find virus, the biological characteristics of the various albumen especially Gn of its genome encoding, the effect during disease development, And amynologic characteristic also knows little about it；Meanwhile, from the point of view of the demand of epidemiology and clinic diagnosis process, need exploitation SFTS phase badly Prevention vaccine, diagnosis antibody and the therapeutic antibodies closed.
Nucleic acid vaccine, also known as DNA vaccination, its mechanism of action mainly has following three kinds: the antigen of DNA encoding passes through somatic cell warp MHC I classpath submission is to CD8+T cell；The antigen presenting cell (such as dendritic cell) that DNA immunization direct transfection is full-time Through MHC I/II classpath by antigen presentation to CTL, CD4+T cell, and activate B cell response；Transfected somatic cell quilt After antigen presenting cell phagocytosis, there is cross activation, by antigen presentation to T cell.It has the advantage that can fully simulate Natural infection state, synthesis has the albumen of additional space conformation in animal body, is possible not only to excitating organism and produces humoral immune reaction Can also the cell immune response of inducing producing specificity；Comparing with recombiant protein immunity, nucleic acid vaccine can be at DNA level to password Son is optimized, modifies, and can preferably ensure the purity of antigen protein, and with low cost, good stability, can avoid egg simultaneously Defect that the white immunogen half-life is short and set up more effective permanent immunity response；Genetic immunization encodes egg by the gene order of amplification Bai Kangyuan, it is possible to avoid direct contact with dangerous higher pathogen.
But nucleic acid vaccine there is also shortcomings, the most most importantly the object of nucleic acid vaccine research and application is mainly eukaryote, And the genes of interest overwhelming majority comes from the prokaryote such as virus or antibacterial, and prokaryote and eukaryote are on codon usage bias There is notable difference, the exogenous gene which results in nucleic acid vaccine can not be expressed in eukaryotic cell efficiently, it is impossible to effectively stimulates The immune system of body produces response.
Summary of the invention
It is an object of the invention to overcome drawbacks described above, it is provided that a kind of codon optimized SFTSV Glycoprotein G n gene order.
Another object of the present invention is to a kind of tPA of carrying signal peptide is provided and carries out the nucleic acid vaccine of codon optimized SFTSV Gn.
The purpose of the present invention is achieved through the following technical solutions:
The original gene sequence of a kind of SFTSV Glycoprotein G n containing wild type signal peptide (WSP), sequence is SEQ ID NO.1.
The gene order of a kind of codon optimized SFTSV Glycoprotein G n, sequence is SEQ ID NO.2.
The nucleic acid vaccine of a kind of SFTSV Glycoprotein G n, is inserted by the gene order of the SFTSV Gn that sequence is SEQ ID NO.1 Gained between carrier for expression of eukaryon Hind III and BamH I restriction enzyme site.
The nucleic acid vaccine of a kind of SFTSV Glycoprotein G n, is inserted into by the SFTSV Gn gene order that sequence is SEQ ID NO.2 Gained between carrier for expression of eukaryon Nhe I and BamH I restriction enzyme site, its 5 ' end connects tPA signal peptide sequence.
Described carrier for expression of eukaryon is pJW4303.
The SFTSV Glycoprotein G n gene order of the genetic modification that the present invention provides and the construction step of nucleic acid vaccine thereof are as follows:
(1) acquisition of the SFTSV Gn original series genetic fragment containing WSP signal peptide
With SFTSV HB29 strain as reference sequences (GenBank:HM745931.1), choose the Glycoprotein G n containing WSP signal peptide Encoding gene (the original series SEQ ID NO.1 before i.e. optimizing), is synthesized by Nanjing Genscript Biotechnology Co., Ltd. and loads load Body pUC57, i.e. pUC57-WSP-Gn.Design primer WSP-Gn-F: CCCAAGCTTATGATGAAAGTCATCTGG(SEQ ID NO.3)；WSP-Gn-R:GAGCTCGGATCCC TAT TACTCAATCCTAACATCATC(SEQ ID NO.4).Expanded by PCR, introduce Hind III and BamH I enzyme action respectively Site.Amplified production Hind III and BamH I double digestion, and reclaim test kit (Omega Agarose Gel DNA with DNA gel Purification Kit, Omega company of the U.S.) reclaim purification purpose fragment, this fragment is the original series containing WSP signal peptide SFTSV Glycoprotein G n gene order, two ends are connected to Hind III and BamH I restriction enzyme site, in order to nucleic acid vaccine The structure of pJW4303-WSP-Gn.
(2) design of codon optimized SFTSV Glycoprotein G n gene order and synthesis
With SFTSV HB29 strain as reference sequences (GenBank:HM745931.1), first choose containing WSP signal peptide gene SFTSV Gn gene, total length 1605bp, then use software OptimumGeneTMAnalyze its gene order, find out its codon Use preference to find out simultaneously and use, from mammalian codons, the codon site that preference is different, substitute with the codon of mammal preference SFTSV Glycoprotein G n gene uses the codon that preference is different, then designs codon optimized SFTSV Gn gene order, Synthesized by Nanjing Genscript Biotechnology Co., Ltd. and load carrier pUC57, i.e. pUC57-WSP-Gn-opt.Codon optimized Protein amino acid sequence coded by gene order keeps consistent with original aminoacid sequence.SFTSV Gn after codon optimized Gene order is SEQ ID NO.2.
(3) carry tPA signal peptide and codon optimized after the acquisition of SFTSV Gn genetic fragment
The recombinant vector pUC57-WSP-Gn-opt containing target sequence that Nanjing Genscript Biotechnology Co., Ltd. is provided, design Primer tPA-Gn-opt-F:GTCACTTCGCTAGCGACAGTGGACCTATTATCTGCG(SEQ ID NO.5)； TPA-Gn-opt-R:GAGCTCGGATCCCTATTACTCAATCCGCACATCGTCC(SEQ ID NO.6).Pass through PCR Amplification, introduces restriction enzyme site Nhe I and BamH I, by Gn-opt gene order even respectively at the 5 ' ends and 3 ' ends optimizing Gn sequence TPA signal peptide sequence downstream at plasmid pJW4303.Amplified production Nhe I and BamH I double digestion, and use DNA gel Reclaim test kit (Omega Agarose Gel DNA Purification Kit, Omega company of the U.S.) and reclaim purification purpose fragment, This fragment is the SFTSV Gn gene order optimized, and two ends are connected to Nhe I and BamH I restriction enzyme site, in order to nucleic acid The structure of vaccine pJW4303-tPA-Gn-opt.
(4) gene fragment clone step (1) obtained is in carrier for expression of eukaryon pJW4303, obtains recombiant plasmid pJW4303-WSP-Gn.Gene fragment clone step (3) obtained, in carrier for expression of eukaryon pJW4303, obtains matter of recombinating Grain pJW4303-tPA-Gn-opt.After to restructuring plasmid extraction, enzyme action, order-checking, determine and obtained plasmid in line, i.e. For SFTSV Glycoprotein G n nucleic acid vaccine of the present invention.
The useful achievement of the present invention:
SFTSV is a kind of newfound pathogen, at present to the biological characteristics of the various albumen especially Gn of its genome encoding, Whether appropriate clinical and epidemiology utilization etc. are the most unclear for effect during disease development, its antibody.Additionally, due to There is the Preference of codon in nature biotechnology body, is difficult to have in the SFTSV Gn gene in pathogen source is cloned in heterologous host body Efficient expression, therefore cannot effectively stimulate the immune system of heterologous host, is allowed to produce preferable immanoprotection action.In order to improve Heterologous gene expression efficiency in mammal, generally requires and is optimized nucleotide coding sequence.Due at present to nucleotides sequence The most unified standard of optimization of row or principle.Therefore for identical aminoacid sequence, different research worker can be designed completely Different nucleotide sequences is used for target polypeptides or the expression of albumen and manufacture, and correspondingly expression efficiency also likely to be present difference.Inventor Based on the SFTSV Gn gene of SFTSV HB29 strain, devise a plurality of codon optimized after SFTSV Gn sequence, and lead to Cross that testing sieve selects that expression efficiency is the highest one.Meanwhile, original for SFTSV Gn signal peptide (WSP) is replaced into tPA by inventor. Compared with wild type gene, introduce and the codon appearance of mammalian cell preference in codon optimized gene through tPA signal peptide Frequency increases, but the SFTSV Gn aminoacid sequence of its coding is constant.Utilize outer-gene restructuring by the gene order of destination protein It is cloned into carrier for expression of eukaryon pJW4303 and constructs nucleic acid vaccine pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt of SFTSV.
By the expression of the technique study Gn of in-vitro transfection, inventor find nucleic acid vaccine pJW4303-WSP-Gn, PJW4303-tPA-Gn-opt all can express in eukaryotic cell 293T cell, but the destination protein of pJW4303-tPA-Gn-opt Gn expression is higher, and Gn can be secreted into 293T extracellular by only pJW4303-tPA-Gn-opt.As can be seen here, phase It is more suitable for compared with the pJW4303-WSP-Gn of wild-type status, the pJW4303-tPA-Gn-opt after our genetic modification Protein expression in mammalian cell.
By the humoral immunogenicity of the technique study Gn of DNA immunization animal, inventor find nucleic acid vaccine pJW4303-WSP-Gn, PJW4303-tPA-Gn-opt all can induce body to produce specific humoral immune response, but pJW4303-tPA-Gn-opt can be faster The Specific antibody titre that ground induction body produces humoral immunoresponse(HI) and generation is higher.As can be seen here, compared to wild-type status PJW4303-WSP-Gn, the pJW4303-tPA-Gn-opt nucleic acid vaccine after our genetic modification has more preferable body fluid and exempts from Epidemic focus.
Accompanying drawing explanation
Fig. 1 wild type and the codon preference comparative result of codon optimized SFTSV Glycoprotein G n gene order
Wherein, Figure 1A and 1B is respectively codon adaptation indexI (CAI) and optimal codon uses frequency, and two figure left part are excellent Result after change, right part is the most optimized wild-type results, SFTSV glycoprotein after the optimization of Figure 1A and 1B prompt cipher Matching degree that the coding region synonymous codon of Gn gene order most preferably uses with codon in mammalian cell and the optimum used are close Numeral ratio significantly improves；Fig. 1 C is the adjustment of G/C content, in prompting SFTSV Glycoprotein G n gene order after codon optimized Base composition has no significant change, it is ensured that uniform annealing temperature during gene chemical synthesis, it is ensured that the expression of mRNA in animal body Level；Fig. 1 D is the optimization of restricted enzyme and cis acting element, and prompting can affect the specific gene sequence of plasmid construction originally By codon optimized effective removing；Fig. 1 E is the optimization of repetitive sequence, and the gene order after prompt cipher sub-optimization can not form stem Ring structure, thus ensure that ribosome and effectively combining of nucleotide sequence and stablizing of mRNA.
Double digestion electrophoretogram after Fig. 2 purpose fragment WSP-Gn, tPA-Gn-opt PCR amplification
A schemes: 1,1kb DNA ladder；2, WSP-Gn through Hind III and BamH I double digestion product；3,100bp DNA ladder；
B schemes: 1, tPA-Gn-opt through Nhe I and BamH I double digestion product；2,100bp DNA ladder；3,1kb DNA ladder.
The restriction enzyme digestion and electrophoresis figure of Fig. 3 pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt
A schemes: 1,1kb DNA ladder；2, pJW4303-WSP-Gn through Hind III and BamH I double digestion product；
B schemes: 1,1kb DNA ladder；2, pJW4303-tPA-Gn-opt through Nhe I and BamH I double digestion product.
After Fig. 4 pJW4303, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt transfection 293T cell, destination protein Gn expresses Western Blot result
1, pJW4303 transfection 293T cell conditioned medium；2, pJW4303 transfection 293T cell pyrolysis liquids；3, pJW4303-WSP-Gn transfections 293T cell conditioned medium；4, pJW4303-WSP-Gn transfection 293T cell pyrolysis liquids；5, pJW4303-tPA-Gn-opt transfection 293T Cell conditioned medium；, 6, pJW4303-tPA-Gn-opt transfection 293T cell pyrolysis liquids.One resists the pJW4303-tPA-Gn-opt for 1:300 dilution Immunity BALB/c mouse serum.
After Fig. 5 pJW4303, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt immunity BALB/c mouse, in serum, specific IgG should Answer time graph
PJW4303 represents the specific IgG antibodies time graph after empty carrier pJW4303 immunity BALB/c mouse, totally 7；
PJW4303-WSP-Gn represents the specific IgG antibodies time graph after pJW4303-WSP-Gn immunity BALB/c mouse, totally 7 Only；
PJW4303-tPA-Gn-opt represents the specific IgG antibodies time graph after pJW4303-tPA-Gn-opt immunity BALB/c mouse, Totally 7.
After Fig. 6 pJW4303, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt immunity BALB/c mouse, the 10th week specific IgG resists Body titre
PJW4303 represents the specific IgG titers after empty carrier immunity BALB/c mouse, totally 7；
PJW4303-WSP-Gn represents the specific IgG titers after pJW4303-WSP-Gn immunity BALB/c mouse, totally 7；
PJW4303-tPA-Gn-opt represents the specific IgG titers after pJW4303-tPA-Gn-opt immunity BALB/c mouse, totally 7.
Detailed description of the invention
The design of the SFTSV nucleoprotein gene sequence that embodiment 1. is codon optimized and synthesis
Use software OptimumGeneTMThe gene order SEQ ID NO.1 of analysis of encoding SFTSV Glycoprotein G n, finds out its password Son uses preference and uses, from mammal, the site that preference is different.For using the codon site that preference is different, use mammal The codon of cell preference substitutes, and design filters out codon optimized SFTSV Glycoprotein G n gene order SEQ ID NO.2. Protein amino acid sequence consensus amino acid sequence original with it coded by above-mentioned codon optimized gene order.Above-mentioned codon The gene order optimized is synthesized by Nanjing Genscript Biotechnology Co., Ltd., loads carrier pUC57, is built into recombiant plasmid pUC57-WSP-Gn-opt.Confirm that through order-checking the sequence of synthesis is correct.
Codon optimized site is carried out in order to be explicitly shown, existing by the nucleotide sequence WSP-Gn-opt after codon optimized and codon Nucleotide sequence WSP-Gn before optimization contrasts.Comparative result is following (* is termination codon):
Above-mentioned codon optimized SFTSV Glycoprotein G n gene order and the sub-Preference of wild-type sequence password comparison there occurs change. By software OptimumGene from Fig. 1TMThe result that simulates is it can be seen that compared with wildtype gene sequence, codon optimized Gene in mammalian cell preference Codon frequencies increase, but they coded aminoacid sequences are constant so that The protein expression that SFTSV Glycoprotein G n is more suitable in mammalian cell.
The structure of embodiment 2. carrier for expression of eukaryon pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt
(1) purpose fragment and the acquisition of carrier
1) WSP-Gn fragment, the acquisition of the linear large fragment of plasmid pJW4303: with pUC57-WSP-Gn as template, use primer WSP -Gn-F(CCCAAGCTTAnd WSP-Gn-R (GAGCTC ATGATGAAAGTCATCTGG)GGATCCCTATTAC TCAATCCTAACATCATC) PCR expands SFTSV Glycoprotein G n gene order, amplified production Hind III and BamH I couple Enzyme action.And with Hind III and BamH I double digestion vector plasmid pJW4303.Endonuclease reaction system is: 10 × Buffer TangoTM 4 μ l, pJW4303 or corresponding PCR primer 10 μ l, Hind III 2 μ l, BamH I 2 μ l, moisturizing to 40 μ l, 37 DEG C, 2h.
2) tPA-Gn-opt fragment, the acquisition of the linear large fragment of plasmid pJW4303: with pUC57-WSP-Gn-opt as template, with drawing Thing tPA-Gn-opt-F (GTCACTTCGCTAGCAnd tPA-Gn-opt-R GACAGTGGACCTATTATCTGCG) (GAGCTCGGATCCCTATTACTCAATCCGCACATCGTCC) the SFTSV sugar egg that PCR amplification is codon optimized White Gn gene order, amplified production Nhe I and BamH I double digestion.And with Nhe I and BamH I double digestion vector plasmid pJW4303.Endonuclease reaction system same 1).
(2) digestion products purification: TAE buffer makes 1% agarose gel, digestion products carries out electrophoresis, 100V, 1h；Weigh Empty 1.5mlEp pipe, cuts the gel containing target DNA and inserts Ep pipe under uviol lamp；Chopping blob of viscose, analytical balance claims the matter of blob of viscose Amount, is carried out calculating the volume of blob of viscose by 100mg=100 μ l；Add at least 1 times of isopyknic Binding Buffer；Mixture is put Melt completely to gel, therebetween every mixing in 2-3 minute once in 55 DEG C～65 DEG C of water-bath middle temperature bath 7min；Shift 700 μ l coolings DNA-agarose solution to one HiBindTM DNA pillar, and pillar is contained in a clean 2ml collecting pipe, under room temperature, 10,000 × g is centrifuged 1min, discards liquid；Pillar is recovered in collecting pipe again, adds 300 μ l Binding Buffer to HiBind In DNA pillar, under room temperature, 10,000 × g is centrifuged 1 minute, discards filter liquor；Pillar is recovered in collecting pipe again, adds In 700 μ l SPW Wash buffer to HiBind DNA pillars, under room temperature, 10,000 × g is centrifuged 1 minute, abandons filter liquor；Weight Washed once again, again recovered in collecting pipe by void column, 10,000 × g is centrifuged the liquid that 1min is remaining to dry base for post matter；? Pillar is contained on a clean 1.5ml centrifuge tube, adds on 30～50 μ l eluents or aquesterilisa on pillar film, 10,000 × g from The heart 1 minute, the solution in centrifuge tube is exactly the DNA product of purification, measures DNA concentration and is stored in-20 DEG C.
(3) coupled reaction: each purpose fragment is connected with the corresponding linear large fragment of plasmid pJW4303 with T4DNA ligase, Obtain pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt recombinant expression plasmid, be SFTSV sugar egg provided by the present invention The nucleic acid vaccine of white Gn.Coupled reaction system is: 10 × T4DNA Ligase Buffer 1 μ l, and linearizing pJW4303 1 μ l is pure The Gn changed is correlated with purpose fragment 7 μ l, T4DNA Ligase 1 μ l, mixing, places 16h for 4 DEG C.Junctional complex converts HB101 competence Cell.
The qualification of embodiment 3. recombiant plasmid pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt
3.1 pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt convert HB101 competent cell respectively
1), under aseptic condition, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt of 5 μ l are added separately to 100 μ l escherichia coli In HB101 competent cell, mix gently, ice bath 30min.
2) the Ep pipe containing cell suspending liquid is placed in 42 DEG C of water-bath thermal shock 90s.
3) after heat-shock treatment, cell is immediately placed on ice 2～3min.
4) add the LB culture fluid 800 μ l without ampicillin, be placed in constant-temperature shaking incubator, 37 DEG C, 100rpm, cultivate 50min。
5) take 0.2ml pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt conversion bacterium solution with pipettor to coat containing 100 μ g/ml ammonia On the agar plate of benzylpcnicillin.
6) being faced up by culture dish and be placed in about room temperature 20min, bacterium solution to be coated is inverted after drying and is put in water isolation type calorstat, 37 DEG C of overnight incubation.
3.2 screening positive clone
Operate according to the Mini Plasmid Purification Kit plasmid Mini Kit description of QIAGEN company.
1) microbionation: picking pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt convert the single colony inoculation on plate in 5ml In LB culture fluid containing 100 μ g/ml ampicillin, 37 DEG C, 180rpm, incubated overnight.
2) being slowly drawn onto in aseptic super-clean bench in 1.5ml centrifuge tube by the little antibacterial shaken overnight, in culture test tube, remaining a small amount of bacterium solution is protected It is stored in 4 DEG C.
3) microorganism collection: bacterium solution in centrifuge tube, under room temperature 13,000g is centrifuged 1min, abandons supernatant.
4) suspension thalline: add 250 μ l buffer P1 abundant suspended bacterial precipitations.
5) alkaline denaturation: add 250 μ l buffer P2, gentle reverse 5-6 time, form clear solution.
6) renaturation: adding the buffer N3 of 400 4 DEG C of pre-coolings of μ l, gentle reverse 5-6 time, then room temperature stands 2min.
7) plasmid separates with albumen, genomic nucleic acids: room temperature 13,000g, centrifugal 10min.
8) absorption of plasmid: Spin Column is placed on Collection Tube, and supernatant in step 7 is joined Spin Column In, 13000g is centrifuged 1min, abandons filtrate.
9) removing protein is fully removed: being added in Spin Column by 500 μ l buffer PB, 13,000g are centrifuged 30s, abandon filtrate.
10) washing: being added in Spin Column by 700 μ l wash buffer, 13,000g are centrifuged 30s, abandon filtrate.
11) repeated washing is once: repeat step 10.
12) being placed on 1.5ml Ep by Spin Column, drip the sterile purified water of 50 μ l in film central authorities, room temperature stands 1min.
13) 13000g is centrifuged 1min, and eluent is the solution containing plasmid.
3.3 enzyme action identify plasmid pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt
PJW4303-WSP-Gn Hind III and BamH I carries out double digestion, pJW4303-tPA-Gn-opt Nhe I and BamH I carries out double digestion, overall reaction system (10 μ l): 10 × Buffer TangoTM1 μ l, plasmid (about 0.20 μ g/ μ l) 2 μ l, Nhe I or Hind III 0.5 μ l, BamH I 0.5 μ l, uses sterilizing ddH2O supplies reaction system to 10 μ l.37 DEG C, hatch 2h, add 1 μ l 10 × Loading buffer terminates endonuclease reaction, 10g/L agarose gel electrophoresis observed result, and Fig. 3 is shown in by enzyme action rear electrophoresis collection of illustrative plates.Fig. 3 The clone of two kinds of nucleic acid vaccines of display all builds correctly.Enzyme action is identified, and correct bacterial clone draws three flat boards continuously, then serves sea Sheng Gong biotech firm checks order, and the monoclonal antibacterial checking order correct is in-80 DEG C of preservations.
Embodiment 4.pJW4303-WSP-Gn, (the big extraction reagent kit of plasmid is QIAGEN in a large amount of preparations of pJW4303-tPA-Gn-opt Plasmid Mega Kit (5), Qiagen company)
1) the antibacterial preservation liquid 10 μ l drawing qualification correct is inoculated in the 5ml LB culture fluid containing 100 μ g/ml ampicillin, 37 DEG C, 180rpm, overnight growth.
2) by 1:500, are cultivated bacterium solution the previous day to be inoculated in the 500ml LB culture fluid containing 100 μ g/ml ampicillin, 37 DEG C, 180rpm, overnight growth.
3) within second day, moving on in 250ml centrifuge bottle by antibacterial, 4 DEG C, 6,000g is centrifuged 15min, abandons supernatant, collects antibacterial.
4) add 50ml buffer P1, repeatedly shake, until antibacterial is all resuspended in solution.
5) adding 50ml buffer P2, gentle reverse 5-6 time, solution, in the bluest, stands 5min.
6) adding 50ml buffer P3, gentle reverse 5-6 time, blue solution disappears, and solution is layered, and upper strata is solid milky group Block, lower floor is clear liquid, places 30min on ice.
7) 4 DEG C, 21,000g are centrifuged 30min, and supernatant is transferred to another centrifuge bottle, again supernatant are centrifuged 10min under the conditions of being somebody's turn to do.
8) take buffer QBT 35ml and balance extraction column.
9) supernatant obtained in (7) is joined in extraction column, naturally cross post, discard filtered solution.
10) add lavation buffer solution QC 200ml, naturally cross post, discard filtered solution.
11) add elution buffer QF 35ml, naturally cross post, collect filtered solution.
12) adding 24.5ml isopropanol in collecting liquid, 4 DEG C, 16,000g are centrifuged 30min, abandon supernatant.
13) precipitating in the resuspended centrifuge tube of 7ml 70% ethanol, 4 DEG C, 16,000g are centrifuged 10min.
14) centrifuge tube having precipitation is dried in super-clean bench naturally, 1ml Elution Buffer dissolution precipitation.
15) plasmid concentration in determined by ultraviolet spectrophotometry extracting gained solution and 260/280 ratio, frozen in-70 DEG C after subpackage.
Embodiment 5. cell transfecting
The 293T cell DMEM high glucose medium containing 100U/ml penicillin, 100 μ g/ml streptomycins and 10% hyclone exists 37 DEG C, 5 CO2Cultivate to exponential phase in saturated humidity incubator, after 2.5g/L trypsinization, with 5.0 × 106Individual cell (6mL) be inoculated in 10cm culture dish, to be grown to 80% merge time, carry out cell transfecting according to PEI infection protocol.Take PEI 75 μ l, PJW4303-WSP-Gn, pJW4303-tPA-Gn-opt 20 μ g, adds containing 100U/ml penicillin, the DMEM of 100 μ g/ml streptomycins High glucose medium, to 825 μ l, fully mixes, incubated at room 15min, is then added in culture dish by above-mentioned mixed liquor and is shaken gently for Make mix homogeneously, simultaneously using pJW4303 empty plasmid transfection 293T cell as negative control.Change only blue or green containing 100U/ml after 8h Mycin, the DMEM culture fluid of 100 μ g/ml streptomycins.Continue to cultivate harvesting lysate after 48h to carry out Western blot and divide Analysis.Wherein, the product of cell lysis results step of transfectional cell is: abandon cell culture supernatant, with PBS (concentration 10mM, pH7.2) By cell eluting from culture dish, collect cell suspension, 2,500rpm, room temperature, centrifugal 10min, abandons supernatant, adds lysate (50mM Tris-HCl PH7.6,150mM NaCl, 1%Triton, add 1%100mM PMSF (Phenylmethanesulfonyl fluoride) before use), ice On hatch 15min, 12.000rpm, 4 DEG C, centrifugal 60min, collect supernatant ,-20 DEG C are frozen.
The vivoexpression of embodiment 6.Western Blot detection pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt
1) preparation of sample: pJW4303, the lysate 20 μ l of each Transfected Recombinant Plasmid 293T cell, adds 5x sample-loading buffer 5 μ l, 100 DEG C, boil 8min.
2) preparative separation glue: 7.5ml 30% acrylamide solution, 3.7ml Tris/Cl PH8.8,150 μ l 10%SDS, 150 μ l 10% Ammonium persulfate., 6 μ l TEMED, encapsulating, above separation gel, add ddH2O fluid-tight, polyase 13 0min under room temperature.
3) preparation concentrates glue: abandon fluid-tight water, the concentration glue of preparation 5% (4.1ml ddH2O, 1ml 30% acrylamide solution, 750 μ l Tris/Cl PH6.8,60 μ l 10%SDS, 60 μ l 10% Ammonium persulfate .s, 6 μ l TEMED), encapsulating, inserts comb, treats that glue is the most solidifying After Ju, pull up comb, glue transfer is fixed in electrophoresis tank.
4) loading: joined by the above-mentioned protein sample handled well and carry out electrophoresis in well, first 20mA 1h, then 40mA continues Continuous electrophoresis 2h.
5) transferring film: methanol activates pvdf membrane, balances in Tris-Glycine buffer, is forwarded to by the albumen on glue with 100V, 1h On pvdf membrane.
6) close: the film taken a turn for the better is closed with 5% defatted milk powder, 37 DEG C, 1h.
7) film is washed twice with PBST.
8) hatch one to resist: be immersed in by film in the BALB/c mouse immune serum of 1:300 dilution, 4 DEG C, overnight incubation.
9) washing film: abandon serum, PBST washes film 6 times, each 10min.
10) hatch two to resist: abandon cleaning mixture, add the sheep anti-mouse igg (1:10.000 dilution) of HPR labelling, 37 DEG C, 1h.
11) abandon two to resist, wash film 6 times with PBST, each 10min.
12) ECL chemical luminous substrate is uniformly added on film, and the development of Bio-Rad imaging system is taken pictures.
Result is shown in Fig. 4, all can be in cell pyrolysis liquid after pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt transfection 293T cell The expression of detection destination protein, but pJW4303-tPA-Gn-opt group destination protein expression is higher and can be secreted into outside born of the same parents, purpose simultaneously Protein G n apparent molecular weight is about 61kDa.Supernatant and the lysate of negative control empty carrier pJW4303 transfection 293T cell are not the most examined Go out the existence of destination protein.
Embodiment 7.pJW4303-WSP-Gn, the research of pJW4303-tPA-Gn-opt nucleic acid vaccine humoral immunogenicity
After nucleic acid vaccine builds and expresses successfully, BALB/c mouse is carried out immunity, detects its immunogenicity by ELISA method. 7.1pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt immunity BALB/c mouse, experimental design is as follows:
Table 1 pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt immunity BALB/c mouse
Such as table 1, by each immune one group of BALB/c mouse of pJW4303, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt.Flesh The 100 corresponding plasmids of μ g injected by meat, carry out internal electrotransfection (pin in injection site WJ-2002 living gene importing equipment immediately after injection Head insertion depth at least 2mm, electrotransfection parameter: voltage 50V, positive and negative each 3 times of pulse number, ripple width 30ms, frequency 30Hz), It is considered as electrotransfection effective with mice leg muscle generation shake.DNA immunization is carried out in the 0th, 2,4,8 weeks, before each immunity, 6th week, final immunization blood sampling two weeks after.
Gn specific IgG in 7.2ELISA detection serum
With IgG antibody reaction special in ELISA method detection serum, evaluate pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt Nucleic acid vaccine induces the ability producing humoral immunization in BALB/c mouse model.
1) antigen coated: pJW4303-tPA-Gn-opt transfection supernatant (uses PBS pH7.2-7.4 as antigen coated elisa plate As diluent), every hole 100 μ l, 4 DEG C, overnight.
2) wash plate: discard and be coated liquid, wash plate 5 times with 0.05%PBST (PBS containing 0.05%Tween-20).
3) close: 1%BSA and 4%Whey (PBS containing 0.05%Tween-20,1%BSA and 4%Whey), every hole 200 μ l, close 1h by 37 DEG C.
4) wash plate: discard confining liquid, wash plate 5 times with 0.05%PBST.
5) sample to be tested is added: adding serum to be detected, i.e. one resists, and initial dilution is 1:200, then carries out 1:2 doubling dilution. Every hole adds 100 μ l, 37 DEG C, hatches 1h (an anti-diluent is the PBS containing 4%whey and 0.5%Tween-20).
6) plate is washed: abandon one and resist, wash plate 5 times with PBST.
7) adding biotin labeled two to resist: biotin labeled sheep anti-mouse igg (1:5,000 dilution), every hole adds 100 μ l, 37 DEG C, Hatch 1h (antibody diluent is the PBS containing 4%whey and 0.5%Tween-20).
8) washing plate: abandon biotin labeled sheep anti-mouse igg, PBST washes plate 5 times.
9) streptavidin of HRP-labelling is added: HRP labelling (1:4,000 dilution), every hole adds 100 μ l, 37 DEG C, hatches 1h (diluent is the PBS containing 4%whey and 0.5%Tween-20).
10) abandoning the streptavidin of HRP labelling, PBST washes plate 5 times.
11) TMB colour developing: TMB solution formula, 1, TMB tablet, 0.1M phosphate/citrate buffer 5ml, double Steam water 5ml, 30% hydrogen peroxide 2 μ l.Every hole adds 100 μ l, left at room temperature 3.5min, and every hole adds the H of 50 μ l 1M2SO4 Color development stopping.
12) microplate reader sets 630nm as reference wavelength, and 450nm is test wavelength, measures and record each hole A450 value, calculates multiple Hole meansigma methods, 2.1 times of sera absorbance value before immunity as cut-off value, and Post-immunisation serum absorbance is less than 0.05 Hole also remove.
BALB/c mouse serum Gn specific IgG response time curve is as shown in Figure 5.pJW4303-WSP-Gn、 PJW4303-tPA-Gn-opt nucleic acid vaccine immunity group mice serum all can detect that Gn specific IgG, and along with the increasing of immune time Adding, immune response strength steps up.The serum of pJW4303 empty carrier group mice fails Gn specific IgG response to be detected.
The BALB/c mouse serum the highest titre of Gn specific IgG antibodies is as shown in Figure 6.Figure shows after the 4th immunity 2 weeks In BALB/c serum, pJW4303-WSP-Gn, pJW4303-tPA-Gn-opt immune group all has the specific antibody of higher titre, PJW4303 empty carrier immune group is then not detected by antibody response.PJW4303-tPA-Gn-opt group and pJW4303-WSP-Gn group, PJW4303 group compares and has significant difference (P < 0.05).PJW4303-WSP-Gn group and pJW4303 group compare and have statistics Learn difference (P < 0.05).
Part that the present invention does not relate to is the most same as the prior art maybe can use prior art to be realized.
Reagent information such as following table involved in embodiment:

Claims (3)

1. codon optimized serious heating companion thrombocytopenic syndromes virus (SFTSV) Glycoprotein G n Gene order, sequence is SEQ ID NO.2.
2. a SFTSV Glycoprotein G n nucleic acid vaccine, it is characterised in that by the SFTSV that sequence is SEQ ID NO.2 Glycoprotein G n gene order, is inserted into gained between carrier for expression of eukaryon Nhe I and BamH I restriction enzyme site, Its 5 ' end connects tPA signal peptide sequence.
SFTSV Glycoprotein G n nucleic acid vaccine the most according to claim 2, it is characterised in that described eucaryon Expression vector is pJW4303.
CN201610324251.4A 2016-05-16 2016-05-16 Codon optimized severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn gene sequence carrying tPA signal peptide and nucleic acid vaccine thereof Pending CN106011155A (en)

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CN111500629A (en) * 2020-04-17 2020-08-07 安徽中盛溯源生物科技有限公司 Method for high expression of laminin-511 variant and application thereof
CN112831523A (en) * 2021-03-16 2021-05-25 厦门宝太生物科技有限公司 SARS-CoV-2-RBD eucaryotic protein expression vector and its preparation method and use
CN113061168A (en) * 2021-03-29 2021-07-02 北京诺思兰德生物技术股份有限公司 Truncated fever with thrombocytopenia syndrome virus Gn protein and application thereof

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US20190231862A1 (en) * 2017-10-16 2019-08-01 SLBiGen, Inc. Gene vaccine for preventing and treating severe fever with thrombocytopenia syndrome
CN108715866A (en) * 2018-05-31 2018-10-30 中国科学院动物研究所 A kind of recombinant viral vector, vaccine and the preparation method and application thereof
CN108715866B (en) * 2018-05-31 2021-02-05 中国科学院动物研究所 Recombinant virus vector, vaccine, preparation method and application thereof
WO2020005028A1 (en) * 2018-06-28 2020-01-02 한국과학기술원 Vaccine composition for preventing or treating diseases caused by severe fever with thrombocytopenia syndrome (sfts) viral infection
CN112399854A (en) * 2018-06-28 2021-02-23 韩国科学技术院 Vaccine composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) viral infection
CN111500629A (en) * 2020-04-17 2020-08-07 安徽中盛溯源生物科技有限公司 Method for high expression of laminin-511 variant and application thereof
CN111500629B (en) * 2020-04-17 2021-11-19 安徽中盛溯源生物科技有限公司 Method for high expression of laminin-511 variant and application thereof
CN112831523A (en) * 2021-03-16 2021-05-25 厦门宝太生物科技有限公司 SARS-CoV-2-RBD eucaryotic protein expression vector and its preparation method and use
CN113061168A (en) * 2021-03-29 2021-07-02 北京诺思兰德生物技术股份有限公司 Truncated fever with thrombocytopenia syndrome virus Gn protein and application thereof
CN113061168B (en) * 2021-03-29 2021-11-09 北京诺思兰德生物技术股份有限公司 Truncated fever with thrombocytopenia syndrome virus Gn protein and application thereof

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