CN106967691A - A kind of recombinant rabies virus for carrying IL-6 gene and its application - Google Patents

A kind of recombinant rabies virus for carrying IL-6 gene and its application Download PDF

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CN106967691A
CN106967691A CN201710087252.6A CN201710087252A CN106967691A CN 106967691 A CN106967691 A CN 106967691A CN 201710087252 A CN201710087252 A CN 201710087252A CN 106967691 A CN106967691 A CN 106967691A
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cail6
recombinant
rhep
virus
rabies virus
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郭霄峰
吴玉婷
罗均
张博越
田钦
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South China Agricultural University
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South China Agricultural University
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a kind of recombinant rabies virus rHEP CaIL6 for carrying Immune-enhancing effect factor IL-6 gene and its application.The recombinant virus is, for skeleton, between G the and L genes that the IL6 genes of dog are inserted to HEP Flury, to obtain recombinant plasmid pHEP CaIL6 with Flury plants of hydrophobin HEP, and finally rescue screening obtains recombinant rabies virus strain rHEP CaIL6.The recombinant virus carries the Immune-enhancing effect factor, can strengthen immune response, and induction produces higher anti-rabies virus neutralizing antibody, preferably the attack of protection body resistance lethal rabies poison；Neutralizing antibody with protection can be produced with relatively low-dose, reduce the cost of Canine vaccine.Moreover, the present invention by IL6 genetic recombination into rabies viruses, can stablize expression IL6 albumen, be avoided that again its over-express, overcome the defect that excessive IL6 causes pathology damage.

Description

A kind of recombinant rabies virus for carrying IL-6 gene and its application
Technical field
The invention belongs to molecular biosciences immunological technique field.It is white that the Immune-enhancing effect factor is carried more particularly, to one kind The recombinant rabies virus of the gene of interleukin 6 and its application.
Background technology
The rabies (Rabies) caused by hydrophobin (Rabies virus, RV) are to infect central nervous system With the characteristics of strong Arbo infectious disease, once morbidity fatal rate almost be up to 100%.Asia and Africa area belongs to rabic height Hair, the domestic animal such as cat, dog is the main host for carrying RV, and about 99% patient is after these animal bites or being scratched Illness, rabies are still the Important Infectious Diseases for seriously threatening these regional people's life securities.China is rabic district occurred frequently, Number of the infected is only second to India, is in second place of the world.With the increase of domestic pets, China it is rabic control and prevent and treat according to So shoulder heavy responsibilities.At present for rabies, mainly based on effective vaccine control, people or animal once fall ill, no special efficacy Medicine can be controlled, so, safely and effectively vaccine is to eliminate China's rabic important means in the human world.
In China, dog only turns into the malicious host of rabic main band and propagating source, wants to eliminate human rabies in China, just The quantity of vagrant dog must be reduced, the immune of domestic dog is popularized, its immunization rate is reached more than 70%, is first eliminated in animal Rabies.Although China has just ratified the inactivation rabies vaccine for animals that several moneys have independent intellectual property right at present, because production-scale Limitation, the inertia higher and that import inactivated vaccine is used for a long time of production cost, domestic inactivated vaccine for animals are not yet big at home Amount is applied.The still preferred import inactivated vaccine in the economically developed city of the overwhelming majority, these inactivated vaccines are for rural area and economy Price is too high for backward areas, it is difficult to which large-scale promotion is used, and domestic inactivated vaccine is although cheap, but its immune effect Fruit is not good.Therefore, it is necessary to further reduce R＆D costs, develop cheap, safe and effective people and use and mad dog for animals Disease vaccine.
Immune-enhancing effect factor interleukin 6 (IL6) has been widely used for the adjuvant of vaccine.IL6 has as molecule adjuvant Various biological function.IL6 first can activate B cell and the maturation of inducing T cell, and enhancing body produces specific antibody Ability；Secondly, IL6 can improve the compatibility of Immunoglobulin IgG, make the ratio of potent antibodies higher, it is significantly more efficient in With antigen or removing cause of disease；Meanwhile, IL6 can also promote the generation of cytotoxic T cell, strengthen cellular immunity, clear in time Except cause of disease；In addition, IL6 is also a kind of stronger inflammatory factor, local inflammation reaction can be induced, appropriate inflammatory reaction is favourable Cause of disease is removed in body and produces stronger immune response.So, using IL6 as molecule adjuvant, it can effectively improve vaccine Immune effect, strengthens the specific immune response of body.But, IL6 is as a kind of inflammatory factor, and overexpression or use can Cause excessive inflammatory reaction and damage body.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming above-mentioned prior art, is learned to do using molecular biosciences Section, using rabies vacciness Candidate Strain HEP-Flury as skeleton, HEP-Flury is inserted by Immune-enhancing effect factor IL6 genes；Pass through Rescue obtains the recombinant rabies strain for carrying dog IL6, being capable of the higher anti-rabies virus neutralizing antibody of immune induction generation Level, reduces the cost of Canine vaccine.Moreover, the present invention by IL6 genetic recombination into rabies viruses, can stablize express IL6 albumen, is avoided that it is over-expressed, overcomes the defect that excessive IL6 causes pathology damage again.
It is an object of the invention to provide a kind of recombinant rabies virus rHEP-CaIL6 for carrying IL-6 gene.
The present invention another object is that it is described carry IL-6 gene recombinant rabies virus rHEP-CaIL6 as or Prepare the application in terms of rabies vacciness.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of recombinant rabies virus rHEP-CaIL6 for carrying dog IL6 genes, is with HEP-Flury plants of hydrophobin For skeleton, the dog IL6 genes shown in SEQ ID NO.1 are inserted into HEP-Flury, obtain carrying the recombinant plasmid of dog IL6 genes PHEP-CaIL6, finally rescue screening obtains recombinant rabies virus strain rHEP-CaIL6.
Wherein, the dog IL6 genes shown in SEQ ID NO.1 are utilized by bioinformatics, from China's mass rearing Expand and obtain in Chinese rural area dog, its IL6 albumen expressed only is acted to almost all of dog.
Specifically：The recombinant rabies virus rHEP-CaIL6 is by dog IL6 gene clonings to hydrophobin HEP- In Flury plants of full length cDNA clones, the recombinant cDNA clone pHEP-CaIL6 of expression dog IL6 albumen is constructed；Then by cDNA The helper plasmid cotransfection BHK-21 cells of pHEP-CaIL6 and expression hydrophobin N, P, G and L albumen are cloned, rescue is obtained Carry one plant of chimeric rabies virus rHEP-CaIL6 of dog IL6 genes.
More specifically, in the recombinant rabies virus rHEP-CaIL6, dog IL6 genes are cloned into hydrophobin strain In the middle of HEP-Flury pseudogenes area, i.e. G genes and L genes, construction strategy is as shown in Figure 1.
Recombinant rabies virus rHEP-CaIL6 carry Immune-enhancing effect factor IL6 albumen, can immune induction produce more High anti-rabies virus neutralizing antibody, and more preferable protecting effect is produced, reduce the cost of Canine vaccine.
There are multiple restriction enzyme sites in the HEP-Flury pseudogenes area that the present invention is saved, it is easy to clone and express multiple external source bases Cause, it is easy to by IL6 gene clonings to hydrophobin strain HEP-Flury.It is according to the present invention that IL6 gene clonings is sick to rabies Malicious pseudogene area carries out the strategy of independent expression, and dog IL6 albumen is by single expression, and non-fusion expression.
As it is a kind of it is preferred can embodiment, recombinant rabies virus rHEP-CaIL6 of the present invention construction method Comprise the following steps：
S1. construction recombination plasmid pHEP-CaIL6：By dog IL6 gene clonings shown in SEQ ID NO.1 to hydrophobin In HEP-Flury plants of full-length cDNAs；
S2. carry out rescue using recombinant plasmid and obtain recombinant virus rHEP-CaIL6.
Preferably, the specific method of step S1 construction recombination plasmids is as follows：
S11. using primer I L6-F/IL6-R amplification dog IL6 genes, and Bsi WI and Pst I restriction enzyme sites are introduced；It is described Primer I L6-F/IL6-R sequence is respectively as shown in SEQ ID NO.2 and 3；
S12. nucleic acid restriction endonuclease Bsi WI and Pst I are utilized, IL6 genes and pHEP-3.0 plasmid pairs are entered respectively Row digestion is handled, and both digestion products connections obtain recombinant plasmid pHEP-CaIL6.
Preferably, pHEP-3.0 plasmids described in step S12 are the recombinant plasmids for carrying HEP-Flury strain full-length cDNAs.
Preferably, the PCR system expanded described in step S11 is：The μ L of template (gene plasmid containing IL6) 0.5,5 × HF The 4 μ L of μ L, dNTP (2.5mM) of Phusion Buffer 10, the μ L of sense primer (10 μM) 2, the μ L of anti-sense primer (10 μM) 2, Phusion high-fidelities enzyme 0.5 μ L, ddH2O 31μL。
Preferably, the PCR reaction conditions expanded described in step S11 are：98℃2min；98 DEG C of 10s, 62 DEG C of 20s, 72 DEG C 1min, runs 30 circulations；Most extend 10min after 72 DEG C.
Preferably, the digestion system of digestion processing is described in step S12：IL6/pHEP-3.0 4μL、10×FD Buffer 2μL、Bsi WI 1μL、Pst I 1μL、ddH2O 13μL。
Preferably, the reaction system connected described in step S12 is：IL6 genes digestion products 7 μ L, pHEP-3.0 digestion is produced The μ L of thing 1, the μ L of 10 × T4 ligases Buffer, 1 μ L, T4DNA ligases 1.
Preferably, step S2 specific method is：
S21. transfect：
S211. BHK-21 cells are cultivated in Tissue Culture Plate；
S212. according to 1：0.25：0.125：0.05：0.075 mass ratio, by recombinant plasmid pHEP-CaIL6, and auxiliary Plasmid pH-N, pH-P, pH-L, pH-G are mixed, and add sterile deionized water, are added and are shaken on transfection reagent, whirlpool oscillator, room After temperature is stood, addition mixes to obtain lysate containing 10% hyclone and DMEM dual anti-100IU/mL；
S213. lysate adds 37 DEG C of 2.5~3h of culture of cell, and backup plate wall gently sucks DMEM, washed carefully with 1 × PBS Once, addition is containing 10% hyclone and dual anti-DMEM again by born of the same parents；In 37 DEG C of 5%CO248h, cell are cultivated in incubator 95% cover with after be transferred to 34 DEG C culture 96h；
S22. virus screening.
Wherein it is preferred to, sterile deionized water and recombinant plasmid pHEP-CaIL6 volume mass ratio are in step S212 75μL/μg；Sterile deionized water and the volume ratio of transfection reagent are 10：1.
Preferably, containing 10% hyclone and DMEM dual anti-100IU/mL and recombinant plasmid pHEP- in step S212 CaIL6 volume mass ratio is 400 μ L/ μ g.
Preferably, 10s is shaken in step S212, is stored at room temperature 20min；
Preferably, step S211 is specifically：To be cultivated in Tissue Culture Plate to cell density is 1 × 105Individual/hole, culture Contain 10% hyclone in liquid；37 DEG C of 12~16h of culture, converge to cell 60%~80%；Washed carefully with 1 × PBS during transfection Born of the same parents are once.
Preferably, the specific method of virus screening is as follows described in step S22：
S221. above-mentioned cell plates are encased with masking foil, -70 DEG C of multigelation cells 3 times, cooling time 1h, room-temperature dissolution； Take supernatant and fresh BHK-21 mixing with cells is in 96 orifice plates, cultivate 48h, 34 DEG C of culture 96h at 37 DEG C thereafter；
S222. incline cell culture fluid, the acetone of 80% precooling is filled it up with per hole, quickly incline acetone, and acetone is filled it up with again, Masking foil encases culture plate, puts -20 DEG C of 30min；Acetone is discarded, sky is dry, and 30 μ L anti-rabies virus N protein fluorescence are added per hole Antibody, 37 DEG C of incubation 1h, discards lysate, cell is washed with 1 × PBS twice, each 5min, then uses deionized water quick wash Cell once, is spontaneously dried.
Fluorescence microscopy Microscopic observation, see a large amount of green fluorescence spots for the positive, that is, save successfully.Save the disease obtained Poison is named as rHEP-CaIL6.
Wherein it is preferred to, the volume ratio of supernatant and fresh BHK-21 cells is 2 in step S221：5；It is described fresh BHK-21 cells cell density be 4 × 105Individual/mL.
In addition, above-mentioned recombinant rabies virus rHEP-CaIL6 as or prepare rabies vacciness in terms of application, Within protection scope of the present invention.
The present invention is first using rabies virus vaccine strain HEP-Flury as skeleton, with the China of the current mass rearing of China The IL6 genes of rural area dog are connected as the Immune-enhancing effect factor by digestion, are built and are contained dog IL6 genes (see SEQ ID NO.1) Recombinant rabies virus rHEP-CaIL6, construction strategy is as shown in Figure 1.Then the recombinant rabies virus saved to success Enter performing PCR, sequencing, identified by immunofluorescence correct.The present invention also carries out growth curve, pathogenic, immunogenicity to recombinant virus With attack the biological characteristic researches such as malicious protection, to assess it as the effect of recombinant vaccine.As a result show, recombinate mad dog Sick virus rHEP-CaIL6 is pathogenic relatively low as recombination engineered vaccine, will not be to, drop on NA cell lethal into mouse Degree is higher than parent plant HEP-Flury, and recombinant virus has good immunogenicity, and body can be induced to produce with Vaccine effectiveness Rabies neutralizing antibody, can preferably protect body to resist the attack of lethal rabies poison.
Specifically in terms of research method, the present invention has infection using molecular biology method structure carrying dog IL6 genes Property total length rabies viruses cDNA, the recombinant rabies poison of expression dog IL6 albumen is saved out by cellular biological technique, using exempting from Epidemiology correlation technique, studies the immunogenicity of recombination engineered vaccine.Carried out in vitro in culture cell and Mice Body Recombinant rabies strain as recombinant vaccine recruitment evaluation.
The present invention is identified the recombinant rabies virus for carrying dog IL6 and passage and RT- on BHK-21 cells PCR is identified.Confirmed through immunofluorescent test, recombinant virus N protein equal successful expression in embedded virus infection cell.Pass through The G-protein and dog IL6 albumen of Western-blot identification recombinant viruses are expressed.By rHEP-CaIL6 in BHK-21 cells Middle passage, by sequencing, does not find that IL6 loses and any mutation.
The present invention is studied growth in vitro curve of the rHEP-CaIL6 recombinant viruses on NA cells.The life of virus Long curve shows that rHEP-CaIL6 growth characteristics compared with parent plant HEP-Flury are basically identical, and rHEP-CaIL6 exists The titre of the 2nd day reaches highest and higher than parent plant.
The present invention is studied pathogenic on mouse of rHEP-CaIL6 recombinant viruses.By rHEP-CaIL6 and parent This plant of HEP-Flury distinguishes intercerebral inoculation mouse, and mouse weight is monitored daily.As a result show, recombinant virus rHEP-CaIL6 and Parent plant HEP-Flury influence mouse weights are basically identical, and the faster quick-recovery body weight of mouse of recombinant virus group, show it Security is higher.
The present invention is studied with poison protection is attacked rHEP-CaIL6 recombinant viruses immunogenicity.By rHEP-CaIL6 with Parent plant HEP-Flury is prepared into the attenuated vaccine of various concentrations respectively, and intramuscular injection is immunized mouse, 7,14, take a blood sample after 21 days Serum is separated, anti-rabies virus neutralizing antibody is detected.As a result show, recombinant rabies virus rHEP-CaIL6 is immunized after mouse The anti-rabies virus neutralizing antibody level higher relative to parent plant that produce, and the recombinant virus of relatively low-dose can be induced RHEP-CaIL6 can produce the neutralizing antibody level more than 0.5IU, and attacking poison strain CVS-11 with rabies standard after 22 days is carried out Protest test, rHEP-CaIL6 immune groups mouse can obtain the survival rate higher than parent plant.
The invention has the advantages that：
Science of the present invention constructs the recombinant rabies virus rHEP-CaIL6 of the expression dog IL6 Immune-enhancing effect factors.Restructuring Hydrophobin has higher virus titer compared to parent plant HEP-Flury, can reduce the cost of Canine vaccine.
The present invention verifies recombinant virus simultaneously can produce the hydrophobin neutralization higher than parent plant in Mice Body Antibody, the recombinant rabies poison of relatively low-dose can produce the neutralizing antibody level more than 0.5IU, that is, reduce immunizing dose, can To reduce the cost of Canine vaccine, new rabies recombinant vaccine Candidate Strain can be used as.
The vaccine effect studies have shown that recombinant rabies poison of the present invention can produce more more preferable than parent plant as attenuated vaccine Protection.Moreover, the present invention by IL6 genetic recombination into rabies viruses, can stablize expression IL6 albumen, its mistake is avoided that again Degree expression, overcomes the defect that excessive IL6 causes pathology damage.
Optimization of the invention by restructuring virus constructs system condition, including rescue condition etc., confirm to recombinate mad dog The Optimal system of sick virus rHEP-CaIL6 structures, can significantly improve success rate, can as rabies viruses reference construct System, especially for generally acknowledged heavy difficult point --- virus rescue.
Brief description of the drawings
Fig. 1 is the genetic recombination strategy for building recombinant rabies virus rHEP-CaIL6.
Fig. 2 is that pHEP-CaIL6 recombinant plasmids PCR identifies electrophoretogram；M：DNA Marker2000,1~2：IL6 PCR expands Increase production thing, 3：Negative control.
Fig. 3 identifies electrophoretogram for the rHEP-CaIL6 recombinant viruses RT-PCR of rescue；M：DNA Marker2000,1~2： IL6 pcr amplification product, 3：Negative control.
Fig. 4 identifies for the rHEP-CaIL6 recombinant viruses direct immunofluorescence of rescue.
Fig. 5 is G-protein and dog IL6 protein expression of the rHEP-CaIL6 recombinant viruses of rescue on NA cells Western blot are identified.
Fig. 6 is growth curve (the * * P of rHEP-CaIL6 recombinant viruses and parent plant HEP-Flury on NA<0.01).
Fig. 7 be rHEP-CaIL6 recombinant viruses on BALB/c mouse body weight influence, while set up parent plant HEP-Flury and Mock control groups.
Fig. 8 is neutralizing antibody in rabies of the recombinant virus rHEP-CaIL6 of carrying dog IL6 genes in KM Mice Bodies Level, while setting up parent plant HEP-Flury and Mock control group (* P<0.05；**P<0.01；***P<0.001；****P< 0.0001)。
Fig. 9 is carries the recombinant virus rHEP-CaIL6 of dog IL6 genes as mad dog after attenuated vaccine immunity mouse 21 days Virus attacks malicious protective rate.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
The amplification of the dog IL6 genes of embodiment 1
1st, from morbidity dead Chinese rural area dog collection inguinal lymph nodes and spleen sample, sample is handled and core Acid is extracted, and carries out RT-PCR amplifications.
(1) primer sequence is shown in Table 1
The IL6 amplimers design of dog, design primer I L6-F/IL6-R (WI containing Bsi and Pst I restriction enzymes position Point and protection base).
Table 1
(2) random primer and Oligo (dT) are utilized, system carries out cDNA synthesis as shown in table 2：
Table 2
(3) cDNA using above-mentioned reverse transcription synthesis enters performing PCR amplification, system is such as template using primer I L6-F/IL6-R Shown in table 3.
Table 3
(4) gel reclaims kit (HiPure Gel Pure DNA Micro Kit) with reference to Magen companies, which is used, says Bright book carries out gel extraction, purifying to PCR primer, is attached with carrier pMD18T.
Coupled reaction system is as shown in table 4：
Table 4
(5) connection product conversion DH5 α competent cells, extract recombinant plasmid, and digestion identification is correct.Sequencing is sent by plasmid, As a result IL6 sequences such as SEQ ID NO.1 are obtained.
The sequence of other dog IL6 genes is downloaded from GenBank, using ClustalX softwares and MEGA6.0 softwares to sequence It is compared, the gene order as a result obtained is the IL6 genes of dog.
The dog IL6 gene clonings of embodiment 2 are to HEP-Flury
1st, construction recombination plasmid pHEP-CaIL6
(1) dog IL6 genes (the sequence such as SEQ obtained in primer I L6-F/IL6-R (being shown in Table 1) amplification embodiments 1 is utilized Shown in ID NO.1), the estimated amplification length of IL6-F/IL6-R primers is 642bp (amplification reaction system is as shown in table 5), is drawn respectively Enter Bsi WI and Pst I restriction enzyme sites.
Table 5
Mentioned reagent is sequentially added in PCR pipe.
PCR reaction conditions are：98℃2min；98 DEG C of 10s, 62 DEG C of 20s, 72 DEG C of 1min, run 30 circulations；Most after 72 DEG C extension 10min.
(3) the IL6 genes for expanding PCR carry out gel extraction, purifying, by the IL6 genes and pHEP-3.0 plasmids of purifying (recombinant plasmid for carrying HEP-Flury strain full-length cDNAs) is handled with Bsi WI and Pst I nucleic acid restriction endonucleases, Digestion system is as shown in table 6：
Table 6
After above-mentioned system is mixed, first 37 DEG C of incubation 3h, are reclaimed with gel reclaims kit.Recovery product is connected, Linked system is as shown in table 7：
Table 7
After above-mentioned system is mixed, as in connection instrument, 16 DEG C of connection 24h, connection product conversion XL10-Gold competence is thin Born of the same parents, screening positive clone extracts recombinant plasmid, and performing PCR Preliminary Identification is entered with primer I L6-F/IL6-R, and testing result is correct, sees Fig. 2.
PCR is identified that correct plasmid send biological (Shanghai) limited company of raw work to carry out sequencing, correctly, plasmid It is named as pHEP-CaIL6.The correct clone of sequencing is gone with reference to amount extracts kit specification in Qiagen companies plasmid Carry pHEP-CaIL6 recombinant plasmids in endotoxin, 4 DEG C preserve and treat that rescue is used.
Embodiment 3 carries out recombinant virus rHEP-CaIL6 rescue and screening using recombinant plasmid pHEP-CaIL6
1st, transfection and viral screening technique are carried out with reference to the method for (2006) such as Inoue (2003) and Guo Xiaofeng.
Transfection：
(1) BHK-21 cells are prepared：12 porocyte culture plates cell densities are 1 × 105Contain 10% in individual/hole, nutrient solution Hyclone；37 DEG C of 12~16h of culture, converge to cell 60%-80%.During transfection cell is washed with 800 μ L1 × PBS once.
(2) ratio of helper plasmid is groped
Recombinant plasmid pHEP-CaIL6 and helper plasmid pH-N, pH-P, pH-L and pH-G to grope ratio as shown in table 8：
Table 8
pHEP-CaIL6 pH-N pH-P pH-L pH-G
1 1.00μg 0.30μg 0.175μg 0.05μg 0.075μg
2 1.00μg 0.30μg 0.155μg 0.07μg 0.075μg
3 1.00μg 0.25μg 0.155μg 0.02μg 0.075μg
4 1.00μg 0.25μg 0.125μg 0.07μg 0.055μg
5 1.00μg 0.25μg 0.125μg 0.05μg 0.075μg
Recombinant plasmid pHEP-CaIL6, and helper plasmid pH-N, pH-P, pH-L and pH-G is taken to mix by the different proportion of table 8 In 1.5mL centrifuge tubes, addition sterile deionized water to 75 μ L, brief centrifugation so that the drop in tube top portion enters in solution.
(3) add on 7.5 μ L transfection reagents, whirlpool oscillator and shake 10s.
(4) 20min is stored at room temperature, DMEMs of the 400 μ L of addition containing 10% hyclone and dual anti-(100IU/mL) is in centrifugation Guan Zhong, turns upside down pipe several times, mixes.
(5) lysate mixed adds cell (hole in 12 well culture plates), and 37 DEG C of 2.5~3h of culture, backup plate wall is light DMEM gently is sucked, cell is washed with 1 × PBS once, DMEM (containing 10% hyclone and dual anti-) is added again.At 37 DEG C 5% CO248h is cultivated in incubator, cell 95% is transferred to 34 DEG C of culture 96h after covering with.
2nd, the screening of rescue recombinant virus plasmid usage ratio and virus screening：
(1) above-mentioned cell plates are encased with masking foil, -70 DEG C of multigelation cells 3 times, cooling time 1h, room-temperature dissolution.Take (cell density is 4 × 10 for 40 μ L of supernatant liquid and the fresh BHK-21 cells of 100 μ L5Individual/mL) 96 orifice plates are mixed in, thereafter 37 DEG C culture 48h, 34 DEG C culture 96h.
(2) incline cell culture fluid, the acetone of 80% precooling is filled it up with per hole, quickly incline acetone, and acetone, tin are filled it up with again Foil paper encases culture plate, puts -20 DEG C of 30min.Acetone is discarded, it is slight empty dry, 30 μ L anti-rabies virus N proteins are added per hole glimmering Photoactivated antibody, 37 DEG C of incubation 1h, discards lysate, cell is washed with 1 × PBS twice, each 5min, then is quickly washed with deionized water Wash cell once.
Spontaneously dry, as a result fluorescence microscopy Microscopic observation shows, when the ratio of plasmid consumption is 1.00 μ g pHEP- CaIL6,0.25 μ g pH-N, can see a large amount of greens glimmering during 0.125 μ g pH-P, 0.05 μ g pH-L and 0.075 μ g pH-G Hot spot point, is as saved successfully.The viral nomenclature that rescue is obtained is rHEP-CaIL6.
The recombinant virus rHEP-CaIL6 of embodiment 4 passage and identification
1st, recombinant virus rHEP-CaIL6 is passed on and cultivated on BHK-21 cells.By the rHEP- of culture CaIL6 carries out RT-PCR identifications, identified by immunofluorescence and G-protein, the detection of IL6 protein expressions.RT-PCR detections utilize primer IL6-F/IL6-R, primer is shown in Table 1.Immunofluorescence is carried out in BHK-21 cells, the malicious N protein of rabies marked using FITC Identification of the antibodies.G-protein and the detection of IL6 protein expressions are carried out in BHK-21 cells, are carried out by referring to Luo etc. (2016) method Western blot are detected.
2nd, RT-PCR testing results are correct, as shown in Figure 3；Show that the recombinant virus rHEP-CaIL6 of the present invention is successively inserted into Dog IL6 genes.Immunofluorescence test result is positive, as shown in figure 4, showing that culture virus is restructuring rabies viruses.Western Blot testing results are correct, as shown in figure 5, showing expression of recombinant virus G-protein and dog IL6 albumen.
Growth in vitro CURVE STUDYs of the recombinant virus rHEP-CaIL6 of embodiment 5 on NA cells
1st, the recombinant virus rHEP-CaIL6 of Secondary Culture is subjected to growth in vitro CURVE STUDY using NA cells.Use cell Nutrient solution adjustment NA cell densities are 2 × 105/ mL, by recombinant rabies poison rHEP-CaIL6 and parent plant HEP-Flury virus Infection concentration is adjusted to after 0.01,37 DEG C of infection 1h of MOI (multiplicity of infection), and abandoning supernatant is added Fresh complete culture solution (10% serum) is in 5%CO2Cultivated in 37 DEG C of incubators, 34 are transferred to when cell converges completely DEG C, co-culture 120h；100 μ L of supernatant nutrient solutions are collected every 24h and direct immunofluorescence antibody test is carried out, when determining different Between virus titer, be depicted as growth curve.
2nd, result is shown, recombinant virus rHEP-CaIL6 reaches highest in 48h titres, and titre is than parent plant HEP- Flury titres are high, see Fig. 6.
The research that the recombinant virus rHEP-CaIL6 of embodiment 6 influences on mouse weight
1st, rHEP-CaIL6 and parent plant HEP-Flury virus titers are diluted to 1 × 10 respectively6FFU/mL, 6~8 weeks The SPF level BALB/c systems female mice in age is divided into 3 groups, i.e. rHEP-CaIL6 groups, HEP-Flury groups and mock groups, every group 8. Every mouse intracranial of experimental group injects 30 μ L virus liquids (about 3.3 × 104FFU/ is only), the μ L DMEM of mock groups intracranial injection 30.In The body weight of every mouse is detected after immune daily, until all mouse weights are stable (about 31 days).By the weight ratio of all mouse Body weight before upper each injection, studies body weight ratio situation of change.
2nd, result is shown, carries the Immune-enhancing effect factor IL6 genetic engineering recombinant virus rHEP-CaIL6 and parent HEP- Flury changes of weight trend is basically identical, and rHEP-CaIL6 groups than HEP-Flury groups earlier start recover body weight, such as Fig. 7.Show that rHEP-CaIL6 has lower pathogenic and higher security.
Immunogenicities of the recombinant virus rHEP-CaIL6 of embodiment 7 in KM Mice Bodies and attack malicious Protective strategy
1st, recombinant rabies virus rHEP-CaIL6 immunogenicities research：
RHEP-CaIL6 and parent plant HEP-Flury virus titers are diluted to 1 × 10 respectively4FFU/mL、1×105FFU/ mL、1×106FFU/mL, the SPF levels Kunming system female mice of 6~8 week old is divided into 7 groups, i.e. rHEP-CaIL6 groups (1 × 104FFU/ mL、1×105FFU/mL、1×106FFU/mL), HEP-Flury groups (1 × 104FFU/mL、1×105FFU/mL、1×106FFU/ ML) and mock groups, every group 10.(injection dosage is about 1 × 10 to the μ L attenuated vaccines of every mouse intramuscular injection of immune group 1003FFU/ Only, 1 × 104FFU/ only, 1 × 105FFU/ is only), the μ L DMEM of mock groups intramuscular injection 100.7th after immune, 14,21 days tail veins adopt Blood prepares serum, and hydrophobin neutralizing antibody detection is carried out using Cliquet etc. (1998) method.
As a result show, recombinant virus can produce the rabies neutralizing antibody higher than parent plant, be all higher than 0.5IU/mL, quilt It is considered with anti-rabies virus protection, and is more than using the recombinant virus rHEP-CaIL6 of relatively low-dose with regard to that can produce 0.5IU/mL neutralizing antibody, is shown in Fig. 8.
2nd, whether there is protection to strong virus attack to verify mouse to obtain after high level immunity, the after immune 22d is inoculated with 50LD to mouse intracranial50The μ L of CVS-11 30, the morbidity of continuous 16d observations mouse and death condition.
As a result show, low dose group rHEP-CaIL6 and HEP-Flury protective rate do not reach protection below 80%, It is required that, middle dose group, rHEP-CaIL6 has reached 80% protective rate, and it is 40% that immune HEP-Flury, which obtains protective rate, high agent Amount group rHEP-CaIL6 has reached 90% protective rate, and it is 60% that immune HEP-Flury, which obtains protective rate,.
The above results show, relative to parent plant HEP-Flury, carry Immune-enhancing effect factor IL6 genetic engineering restructuring Viral rHEP-CaIL6 just can produce higher immune response in relatively low-dose and offer meets the immune dog of international requirement and attacks poison The standard of protective rate more than 80%, is shown in Fig. 9.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>A kind of recombinant rabies virus for carrying IL-6 gene and its application
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 624
<212> DNA
<213>Dog IL6 genes
<400> 1
atgaactccc tctccacaag cgccttctcc ctggggctgc tcctggtgat ggctactgct 60
ttccctaccc cgggacccct ggcaggagat tccaaggatg atgccacttc aaatagtcta 120
ccactcacct ctgcaaacaa agtggaagaa ctgattaagt acatcctcgg caaaatctct 180
gcactgagaa aggagatgtg tgacaagttt aacaagtgtg aagacagcaa agaggcactg 240
gcagaaaata acctacatct tcccaaactg gagggaaaag atggatgctt ccaatctggg 300
ttcaatcagg agacctgctt gacaagaatc actaccggtc ttgtggagtt tcagctacac 360
ctgaatatcc tccagaacaa ctatgaaggt gataaggaaa atgtcaagtc tgtgcacatg 420
agtaccaaga tcctggtcca gatgctaaag agcaaggtaa agaatcagga tgaagtgacc 480
actcctgacc caaccacaga cgccagcctg caggctatct tgcagtcaca ggatgagtgg 540
ctgaagcaca caacaattca cctcatcctg cggagtctgg aggatttcct gcagttcagt 600
ctgagggctg ttcggataat gtag 624
<210> 2
<211> 30
<212> DNA
<213>Primer I L6-F
<400> 2
attcgtacga tgaactccct ctccacaagc 30
<210> 3
<211> 27
<212> DNA
<213>Primer I L6- R
<400> 3
gttctgcagc tacattatcc gaacagc 27

Claims (10)

1. a kind of recombinant rabies virus rHEP-CaIL6 for carrying dog IL6 genes, it is characterised in that be with hydrophobin HEP-Flury plants are skeleton, and the dog IL6 genes shown in SEQ ID NO.1 are inserted into HEP-Flury, obtain carrying dog IL6 genes Recombinant plasmid pHEP-CaIL6, finally rescue screening obtain recombinant rabies virus strain rHEP-CaIL6.
2. recombinant rabies virus rHEP-CaIL6 according to claim 1, it is characterised in that dog IL6 genes are cloned To between HEP-Flury G genes and L genes.
3. recombinant rabies virus rHEP-CaIL6 according to claim 1, it is characterised in that build by the following method Arrive：
S1. construction recombination plasmid pHEP-CaIL6：By dog IL6 gene clonings shown in SEQ ID NO.1 to hydrophobin HEP- In Flury plants of full-length cDNAs；
S2. carry out rescue using recombinant plasmid and obtain recombinant virus rHEP-CaIL6.
4. recombinant rabies virus rHEP-CaIL6 according to claim 3, it is characterised in that step S1 builds restructuring matter The specific method of grain is as follows：
S11. using primer I L6-F/IL6-R amplification dog IL6 genes, and introduceBsi WI andPst I restriction enzyme sites；The primer IL6-F/IL6- R sequence is respectively as shown in SEQ ID NO.2 and 3；
S12. nucleic acid restriction endonuclease is utilizedBsi WI andPst I, carries out enzyme to IL6 genes and pHEP-3.0 plasmid pairs respectively Processing is cut, both digestion products connections obtain recombinant plasmid pHEP-CaIL6.
5. recombinant rabies virus rHEP-CaIL6 according to claim 4, it is characterised in that pHEP- described in step S12 3.0 plasmids are the recombinant plasmids for carrying HEP-Flury strain full-length cDNAs.
6. recombinant rabies virus rHEP-CaIL6 according to claim 3, it is characterised in that step S2 specific method For：
S21. transfect：
S211. BHK-21 cells are cultivated in Tissue Culture Plate；
S212. according to 1：0.25：0.125：0.05：0.075 mass ratio, by recombinant plasmid pHEP-CaIL6, and helper plasmid PH-N, pH-P, pH-L, pH-G are mixed, and add sterile deionized water, are added and are shaken on transfection reagent, whirlpool oscillator, room temperature is quiet Postpone, addition mixes to obtain lysate containing 10% hyclone and DMEM dual anti-100IU/mL；
S213. lysate adds 37 DEG C of 2.5 ~ 3h of culture of cell, and backup plate wall gently sucks DMEM, cell one is washed with 1 × PBS Secondary, addition is containing 10% hyclone and dual anti-DMEM again；In 37 DEG C of 5%CO248h is cultivated in incubator, cell 95% is covered with After be transferred to 34 DEG C culture 96h；
S22. virus screening.
7. recombinant rabies virus rHEP-CaIL6 according to claim 6, it is characterised in that sterilizing is gone in step S212 Ionized water and recombinant plasmid pHEP-CaIL6 volume mass ratio are 75 μ L/ μ g；The volume of sterile deionized water and transfection reagent Than for 10：1.
8. recombinant rabies virus rHEP-CaIL6 according to claim 6, it is characterised in that contain 10% in step S212 Hyclone is 400 μ L/ μ g with DMEM dual anti-100IU/mL and recombinant plasmid pHEP-CaIL6 volume mass ratio.
9. recombinant rabies virus rHEP-CaIL6 according to claim 6, it is characterised in that shaken in step S212 10s, is stored at room temperature 20min；
Step S211 is specifically：To be cultivated in Tissue Culture Plate to cell density is 1 × 105Contain 10% tire ox in individual/hole, nutrient solution Serum；37 DEG C of 12 ~ 16h of culture, converge to cell 60% ~ 80%；During transfection cell is washed with 1 × PBS once.
10. any recombinant rabies virus rHEP-CaIL6 of claim 1~9 as or prepare rabies vacciness in terms of Application.
CN201710087252.6A 2017-02-17 2017-02-17 A kind of recombinant rabies virus for carrying IL-6 gene and its application Pending CN106967691A (en)

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CN109022375A (en) * 2018-08-21 2018-12-18 山东大学 A kind of recombinant rabies virus that expressing SFTSV Gn albumen and its construction method and application
CN113376380A (en) * 2021-05-31 2021-09-10 华南农业大学 ELISA kit for detecting dog IL-6 and application thereof
CN114805537A (en) * 2022-04-26 2022-07-29 华南农业大学 Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109022375A (en) * 2018-08-21 2018-12-18 山东大学 A kind of recombinant rabies virus that expressing SFTSV Gn albumen and its construction method and application
CN113376380A (en) * 2021-05-31 2021-09-10 华南农业大学 ELISA kit for detecting dog IL-6 and application thereof
CN114805537A (en) * 2022-04-26 2022-07-29 华南农业大学 Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof

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