CN1442479A - Mammalian engineering cell line for production of hantan virus granules - Google Patents
Mammalian engineering cell line for production of hantan virus granules Download PDFInfo
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Abstract
A group of engineering cell systems of mammal for preparing the hantavirus-like particles is disclosed, which integrates the M and S genes or only M gene of HTN-type, SEO-type, PUU-type and DOB-type hantaviruses. The viral glucoproteins G1 and G2 coded by said M gene and the viral nudeoprotein coded by M gene can be stably expressed to form their hantavirus-like particles, which can be used as the genetically engineered vaccine for preventing HFRS, diagnosing preparation, and trangenic carrier.
Description
Invention field
The invention belongs to virusology, molecular biology and field of biological product, relevant with virus vaccines, virus disease diagnosis and gene therapy.
Background of invention
Hantaan virus (Hantanvirus) Hantaan virus (Bunyaviridae), be segmented minus-stranded rna virus, genome is by big (large, L), (medium in, M), little (Small, S) 3 RNA fragments are formed RNA polymerase, glycoprotein g1 and G2, the nucleoprotein of the dependenc RNA of encoding respectively.Hantaan virus extensively distributes in the whole world, can cause very serious disease behind the infection human body, and as hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS) etc., hemorrhagic fever with renal syndrome is called epidemic hemorrhagic fever again in China.China is subjected to the most serious country of Hantaan virus harm, and the hemorrhagic fever with renal syndrome case of the whole world more than 90% occurs in China.Up to the present, in 32 provinces, cities and autonomous regions of China mainland, there are 29 to find Hantaan virus or hemorrhagic fever with renal syndrome case.Therefore, it is especially significant to carry out the Hantaan virus correlative study in China.
At present, the Hantaan virus inactivated vaccine that is using mainly contains 3 kinds: the mouse brain inactivated vaccine of hamster kidney cell inactivated vaccine, gerbil jird nephrocyte inactivated vaccine, purifying.These traditional vaccine productions are used all is that the method for primary cell culture or animal organ's inoculation is cultivated live virus, re-uses to make behind the inactivator inactivation of viruses.Because therefore primary cell or animal organ's itself unhomogeneity has output and the unsettled shortcoming of quality.Since must use the inactivator deactivation, can be influential to virus antigenicity.Must use live virus in the production of inactivated vaccine, exist the infected and environmental pollution problems of staff.Inactivator formalin that uses in the production of vaccine or beta-propiolactone all are carcinogens.The purpose of development of new vaccine then is to avoid these problems.
Vaccinia virus is as the existing many successful examples of vector expression Hantaan virus structural protein.United States Army transmissible disease institute is a carrier with the vaccinia virus, has expressed the M and the S fragment of Hantaan virus 76-118 strain, has made up single expression M and S and has expressed the vaccinia virus recombinant that M adds S simultaneously, and prepared vaccine with the vaccinia virus recombinant of coexpression M and S.But because the shortcoming that has of vaccinia virus vector itself,, make it to be difficult to widespread use as serious side reaction etc.With the baculovirus is that vector expression total length M fragment, total length S fragment and G1 and G2 subunit also succeed, but at present from this expression system purified virus albumen still have masses of work to do to the vaccine for man level.Before the present invention occurred, Shang Weijian had the report that successfully obtains stably express Hantaan virus sample particulate mammal cell line.
Develop a kind of convenience, reliably the serum diagnosis method detects the diagnosis that the intraserous anti-hantavirus antibody of infected individual not only helps virus disease, and can be used for other evaluation of virus type by the antibody that detects type specificity, this all has great importance for clinical and epidemiology.Verified, some epitopes on the Hantaan virus glycoprotein and conformation height correlation owing to can not translate post-treatment, have been lost the antigenicity and the immunogenicity of some natural virals at the glycoprotein of expression in escherichia coli.This nucleoprotein of expressing in eukaryotic cell provided by the invention and glycoprotein can be used as more representative antigen or more effective immunogen.
The reality that the present Research that infects based on current Hantaan virus and CHO (Chinese hamster ovary cell) cell, Vero cell (African green monkey kidney cell) have been used for production of vaccine, it will be very useful setting up by these two kinds of mammal cell line express recombinant Hantaan virus structural protein and reorganization Hantaan virus sample particulate method.The immunological characteristic that reorganization Hantaan virus structural protein that this system produces and Hantaan virus sample particle have native protein, this recombinant protein with native conformation, heavy virus-like particle and their various combination are very useful for prevention, diagnosis and the treatment of Hantaan virus disease.
Detailed Description Of The Invention
Produce Hantaan virus sample particulate Mammals engineering cell system and uses thereof for one group of the present invention's statement, comprise that mainly this Hantaan virus sample particle has and similar immunogenicity of natural Hantaan virus and antigenicity by four kinds of Hantaan virus type M that cause renal syndrome-hemorrhagic fever (HFRS) and the different Hantaan virus types that produced after the S gene integration is gone into cells of mamma animals and the virus-like particle and the production clone thereof of genomic constitution.Its main application will comprise unit price and polyvalent vaccine as the novel Hantaan virus recombinant vaccine of a class.The clone of being stated will be used for the production of novel Hantaan virus recombinant vaccine, and the vaccine of being produced will be used for the prevention of HFRS, can be used for the research and development of the novel carriers in diagnostic reagent and the gene therapy simultaneously.
Produce Hantaan virus sample particulate Mammals engineering cell system and uses thereof for one group of the present invention's statement, main contents comprise following some: 1, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: every kind of engineering cell system is integrated with M segment and the S segment genome or the single M segment genome of above-mentioned various Hantaan virus of HTN (HTN), soul type (SEO), palm fibre back of the body flounder type (PUU), four serotype hantaan types of Du's Bulova type (DOB) virus respectively.2, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: engineering cell system is divided into two classes, but the Hantaan virus nucleoprotein that the Hantaan virus glycoprotein g1 of a class various hantaan type virus M segment genome coding that be stably express and G2 and S segment genome are encoded; Another kind of Hantaan virus glycoprotein g1 and the G2 that expresses various hantaan type virus M segment genome coding for single stable.3, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: common continual and steady the expression or the single continual and steady expression of Hantaan virus glycoprotein in mammalian cell in mammalian cell by Hantaan virus glycoprotein and nucleoprotein, can in cell, form Hantaan virus sample particle justacrine to the extracellular, thus in cells and supernatant sustainable results Hantaan virus sample particle.4, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: the Hantaan virus sample particle that any type integrator gene described in this group clone Sustainable Production claim (1) and (2) in the four type Hantaan virus is determined, comprising the virus-like particle of Hantaan virus glycoprotein and the formation of nucleoprotein co expression and the virus-like particle of the single expression formation of Hantaan virus glycoprotein.5, claim (1-4) is produced Hantaan virus sample particulate Mammals engineering cell system for described one group, it is characterized by: this cell is Chinese hamster ovary cell (Chinese hamster ovary celI) and African green monkey kidney cell (Vero cell), also can be any possible mammal cell that is used to produce.6, according to claim (4), produce the Hantaan virus sample particle that different Hantaan virus types that Hantaan virus sample particulate Mammals engineering cell system produced and different genes are formed, can use in external independent or any compatible combination for one group according to the needs of practical application from now on.7, produce Hantaan virus sample particulate Mammals engineering cell system for one group; it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed; have with the glycoprotein of natural Hantaan virus and nucleoproteide like immunogenicity; no matter be that single type reorganization Hantaan virus sample particle or different type Hantaan virus sample particle compatibility use, all can inducing producing specificity neutralizing antibody and other corresponding immune responses in animal or human's body.8, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed, have with the glycoprotein of natural Hantaan virus and nucleoproteide like antigenic characteristic, can induce the polyclone of generation and monoclonal antibody generation specificity to combine with natural Hantaan virus structural protein.9, according to claim (1-8); one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system; it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed; can be used to prepare the Hantaan virus vaccine; can prepare the Hantaan virus vaccine with single type Hantaan virus sample particle or different type Hantaan virus sample particulate various combination; after these vaccines use in the crowd, can prevent other Hantaan virus of different shaped to infect.9, according to claim (1-8); one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system is characterized in that: one group of Hantaan virus reorganization nucleoprotein, recombinant glycoprotein, virus-like particle or their combination of expressing with mammal cell line constitutes Hantaan virus diagnostic kit and Hantaan virus classification diagnosis test kit as antigen component.10, according to claim (1-8), one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system, it is characterized in that: by various molecular biology methods, the gene that has therapeutic action in Hantaan virus sample granule interior packing, these genes can be RNA or DNA, by Hantaan virus sample particle with these transgenosis in body, as the transfer vector of gene therapy.So far the Hantaan virus recombinant vaccine of domestic and international any form not success as yet.Producing Hantaan virus sample particulate Mammals engineering cell system and uses thereof for one group of the present invention's statement, is invention first in the world, and the development of viral genetic engineering vaccine and the equal tool of prevention of virus disease are had very important significance.The technology of preparing of virus sample particle vaccines not only can be used for the preparation of Hantaan virus sample particle vaccines, also can be used for the preparation of other virus sample particle vaccines.
Following preferential embodiment elaborates to the present invention, and load does not mean that restriction content of the present invention.In these embodiments, be explanation the present invention, the virus strain of be used to increase various Hantaan virus M segment and S segment genome is preserved for this laboratory, and expression vector is by professor's Liang Mifang structure, number of patent application: 01102214.0.Used Chinese hamster ovary celI derives from the hereditary chamber of this institute.Embodiment 1-4 is that one group of production Hantaan virus sample particulate Mammals engineering cell is the preparation method; Embodiment 5-12 is one group and produces Hantaan virus sample particulate Mammals engineering cell system and product applicability feature thereof.
The Construction of eukaryotic that embodiment 1 comprises Hantaan virus glycoprotein gene coding region sequence is with the coding region sequence of PCR method amplification Hantaan virus A9 strain M fragment from the initiator codon to the terminator codon, add the PmlI restriction enzyme site in amplification with upstream primer 5 ' end, 5 ' end at downstream primer adds the XbaI enzyme cutting site, behind PCR product usefulness PmacI and XbaI enzyme cutting, the agarose gel electrophoresis purifying enzyme is cut product.Perhaps with the TA cloning process PCR product is connected the T carrier, after screening has the clone that the PCR product inserts, extract plasmid, with PmacI and XbaI enzymic digestion, the agarose gel electrophoresis purifying enzyme is cut product.Behind plasmid pEF usefulness PmacI and XbaI enzyme cutting, the agarose gel electrophoresis purifying enzyme is cut vector plasmid.To be connected with the carrier that enzyme is cut the back purifying by the A9M fragment coding region sequence of purifying after enzyme is cut, transformed into escherichia coli has the clone of insertion with PCR or the screening of enzyme blanking method.The correct gram called after pEF-A9M that inserts, in pEF-A9M, 5 ' end upstream of A9M fragment sequence is the EF promotor, 3 ' end downstream is a BGH poly adenosine signal sequence.
The Construction of eukaryotic that embodiment 2 comprises Hantaan virus nucleoprotein gene coding region sequence is with the coding region sequence of PCR method amplification Hantaan virus A9 strain S fragment from the initiator codon to the terminator codon, add the PmlI restriction enzyme site in amplification with upstream primer 5 ' end, 5 ' end at downstream primer adds the XbaI enzyme cutting site, behind PCR product usefulness PmacI and XbaI enzyme cutting, the agarose gel electrophoresis purifying enzyme is cut product.Perhaps with the TA cloning process PCR product is connected the T carrier, after screening had the clone of insertion, behind PmacI and XbaI enzyme cutting, the agarose gel electrophoresis purifying enzyme was cut product.Behind plasmid pEF1 α usefulness PmacI and XbaI enzyme cutting, the agarose gel electrophoresis purifying enzyme is cut vector plasmid.To be connected with the carrier that enzyme is cut the back purifying by the A9S fragment coding region sequence of purifying after enzyme is cut, transformed into escherichia coli has the clone of insertion with PCR or the screening of enzyme blanking method.The correct clone's called after pEF-A9S that inserts, in pEF-A9S, 5 ' end upstream of A9S fragment sequence is the EF promotor, 3 ' end downstream is a BGH poly adenosine signal sequence.
The foundation of the Chinese hamster ovary celI system of embodiment 3 stably express Hantaan virus glycoprotein is in order to obtain the Chinese hamster ovary celI system of stably express Hantaan virus A9 strain glycoprotein, with plasmid pEF-A9M liposome method transfection dhfr
-Chinese hamster ovary celI.Cell after the transfection screens with containing the MTX substratum, screens the cell clone of stably express Hantaan virus glycoprotein by the method that improves constantly drug level.Use monoclonal antibody to detect the expression of Hantaan virus glycoprotein in cell by indirect immunofluorescence method at Hantaan virus glycoprotein.After waiting to obtain to express the Chinese hamster ovary celI clone of Hantaan virus A9 strain glycoprotein, carry out the purifying of cell clone with the limiting dilution method, behind twice purifying, proceed passage, enlarged culturing is put the liquid nitrogen preservation with the engineering cell of different generations.
The foundation of the Chinese hamster ovary celI system of embodiment 4 stably express Hantaan virus glycoprotein and nucleoprotein is in order to obtain to stablize the Chinese hamster ovary celI system of coexpression Hantaan virus A9 strain glycoprotein and nucleoprotein, with plasmid pEF-A9M and pEF-A9S liposome method cotransfection dhfr
-Chinese hamster ovary celI.Cell after the transfection screens with containing the MTX substratum, screens the cell clone of stably express Hantaan virus glycoprotein and nucleoprotein by the method that improves constantly drug level.Use respectively at Hantaan virus glycoprotein and nucleoprotein monoclonal antibody and detect Hantaan virus glycoprotein and the expression of nucleoprotein in cell by indirect immunofluorescence method.After waiting to obtain the Chinese hamster ovary celI clone of coexpression Hantaan virus A9 strain glycoprotein and nucleoprotein, carry out the purifying of cell clone with the limiting dilution method, behind twice purifying, proceed passage, enlarged culturing is put the liquid nitrogen preservation with the engineering cell of different generations.
Embodiment 5 expresses the culture supernatant of the Chinese hamster ovary celI system of Hantaan virus A9 strain glycoprotein by the morphology collection of the virus-like particle that Hantaan virus glycoprotein forms, the centrifugal removal cell debris of 10000g, used BeckmanSW40 rotary head 35000rpm then centrifugal 3 hours, with the resuspended precipitation of PBS.Resuspended precipitation is carried out the wolframic acid negative staining, under electron microscope, observe virus-like particle.Under electron microscope, can see Hantaan virus sample particle with double-deck membrane structure and spinous process.Fig. 1 shows by Hantaan virus glycoprotein form the Hantaan virus sample particle of justacrine in the culture supernatant in the CHO engineering cell
The morphology of the virus-like particle that embodiment 6 is formed by Hantaan virus glycoprotein and nucleoprotein coexpression is collected the culture supernatant of the Chinese hamster ovary celI system of coexpression Hantaan virus A9 strain glycoprotein and nucleoprotein, the centrifugal removal cell debris of 10000g, used BeckmanSW40 rotary head 35000rpm then centrifugal 3 hours, with the resuspended precipitation of PBS.Resuspended precipitation is carried out the wolframic acid negative staining, under electron microscope, observe virus-like particle.Under electron microscope, can see Hantaan virus sample particle with double-deck membrane structure and spinous process.Fig. 2 shows by forming the Hantaan virus sample particle of justacrine in the culture supernatant in Hantaan virus glycoprotein and the nucleoprotein CHO engineering cell jointly
Embodiment 7 prepares 10% polyacrylamide gel by the SDS-PAGE of the virus-like particle that Hantaan virus glycoprotein forms.To add 2 * protein electrophoresis sample-loading buffer of equivalent through the Hantaan virus sample particle 20 μ l that form by glycoprotein of ultracentrifugation purifying, boiling water bath 5 minutes, centrifugal 5 minutes of 10000g gets sample electrophoresis on the supernatant.When treating that tetrabromophenol sulfonphthalein arrives the gel bottom, stop electrophoresis.Gel is with examining the dyeing of Ma Shi light blue, observations after decolouring.Can see molecular weight and be the Hantaan virus glycoprotein g2 band of 55Kd and molecular weight and be the Hantaan virus glycoprotein g1 band about 68Kd.The 2nd swimming lane shows among Fig. 3, contains Hantaan virus glycoprotein g2 band that molecular weight is 55Kd and molecular weight behind the Hantaan virus sample particle electrophoresis that glycoprotein becomes and be the Hantaan virus glycoprotein g1 band about 68Kd.The 3rd swimming lane is the molecular weight of albumen mark among Fig. 3.Embodiment 8 prepares 10% polyacrylamide gel by the SDS-PAGE of the virus-like particle that Hantaan virus glycoprotein and nucleoprotein coexpression form.To add 2 * protein electrophoresis sample-loading buffer of equivalent through the Hantaan virus sample particle 20 μ l that form by glycoprotein and nucleoprotein of ultracentrifugation purifying, boiling water bath 5 minutes, centrifugal 5 minutes of 10000g gets sample electrophoresis on the supernatant.When treating that tetrabromophenol sulfonphthalein arrives the gel bottom, stop electrophoresis.Gel is with examining the dyeing of Ma Shi light blue, observations after decolouring.Can see molecular weight and be the Hantaan virus nucleoprotein band of 50Kd, Hantaan virus glycoprotein g2 band that molecular weight is 55Kd and molecular weight and be the Hantaan virus glycoprotein g1 band about 68Kd.The 1st swimming lane shows among Fig. 3, behind the Hantaan virus sample particle electrophoresis that glycoprotein and nucleoprotein are formed, Hantaan virus glycoprotein g2 band, the molecular weight that contains molecular weight and be 55Kd is that Hantaan virus glycoprotein g1 band about 68Kd and molecular weight are the nucleoprotein band about 50Kd.The 3rd swimming lane is the molecular weight of albumen mark among Fig. 3.
The detection of embodiment 9 Hantaan virus nucleoprotein will be through the Hantaan virus sample particle 20 μ l that are made up of glycoprotein and nucleoprotein of ultracentrifugation purifying, 2 * protein electrophoresis the sample-loading buffer that adds equivalent, boiling water bath 5 minutes, centrifugal 5 minutes of 10000g gets sample electrophoresis on the supernatant.When treating that tetrabromophenol sulfonphthalein arrives the gel bottom, stop electrophoresis.Albumen on the gel by half-dried transfer, is transferred on the nitrocellulose filter.At the skim-milk that contains 10%, incubated at room is 1 hour in 0.02%Tween20 and the Tris damping fluid with film, wash 6 times with PBS-Tween20 after, add 4 ℃ of reactions of Hantaan virus nucleoprotein monoclonal antibody and spend the night; After washing 6 times, inferior daily PBS-Tween20, adds the substrate colour developing after washing 6 times with anti-mouse IgG-HRP ELIAS secondary antibody room temperature 1 hour.Fig. 4 shows that the specific staining band of nucleoprotein appears in virus-like particle that the 1st swimming lane is formed jointly by glycoprotein and nucleoprotein, and the virus-like particle that the 2nd swimming lane is formed separately by glycoprotein does not then have the specific staining band of nucleoprotein.The 3rd swimming lane is the molecular weight of albumen mark.
The reaction of embodiment 10 Hantaan virus sample particles and Hantaan virus antibody detects Hantaan virus sample particulate antigenicity with enzyme linked immunosorbent assay (ELISA).Coated elisa plate after will dilute at 1: 100 through the Hantaan virus sample particle of ultracentrifugation purifying, every hole 100 μ l, 4 ℃ are spent the night.Inferior daily PBS-Tween20 washes plate, the patient's HFRS convalescent phase serum, rabbit anti-hantavirus immune serum, mouse-anti Hantaan virus glycoprotein monoclonal antibody, mouse-anti Hantaan virus nucleoprotein monoclonal antibody, people's anti-hantavirus glycoprotein monoclonal antibody, the people's anti-hantavirus nucleoprotein monoclonal antibody that add suitably dilution respectively, hatch after 1 hour for 37 ℃ and wash plate 6 times, the second antibody that adds corresponding horseradish peroxidase-labeled, hatch after 1 hour for 37 ℃ plate is washed 6 times, add the substrate colour developing.The virus-like particle that table 1 is as seen formed jointly by Hantaan virus glycoprotein and nucleoprotein can with patient's HFRS convalescent phase serum, rabbit anti-hantavirus immune serum, mouse-anti Hantaan virus glycoprotein monoclonal antibody, mouse-anti Hantaan virus nucleoprotein monoclonal antibody, people's anti-hantavirus glycoprotein monoclonal antibody, people's anti-hantavirus nucleoprotein monoclonal antibody reactive, the virus-like particle that forms by Hantaan virus glycoprotein can with patient's HFRS convalescent phase serum, rabbit anti-hantavirus immune serum, mouse-anti Hantaan virus glycoprotein monoclonal antibody, people's anti-hantavirus glycoprotein monoclonal antibody reactive, can not with the monoclonal antibody reactive of anti-hantavirus nucleoprotein.Table 1 is the virus-like particle that formed by Hantaan virus glycoprotein list worm and the result of different antibodies reaction, and table 2 be the result that the virus-like particle that formed jointly by Hantaan virus glycoprotein and nucleoprotein and different antibodies are reacted.
The virus-like particle that embodiment 11 experimentation on animalies are formed jointly by Hantaan virus glycoprotein and nucleoprotein and carry out the rabbit immunization experiment respectively by the virus-like particle that Hantaan virus glycoprotein forms separately.After suitably diluting with Hantaan virus sample particle through the ultracentrifugation purifying and aluminum hydroxide adjuvant mix, immunizator focuses on the rabbit about 2.5 kilograms, every animal back leg intramuscular injection 1ml Hantaan virus sample particle.Every animal blood sampling separation of serum is preserved before the inoculation.Inoculation back is the 14th day first, and every animal is intramuscular injection 1ml Hantaan virus sample particle once more, and every animal blood sampling separation of serum is preserved before the inoculation.Inoculation back blood sampling in the 14th day for the second time, separation of serum is preserved.
Embodiment 12 Hantaan virus sample particles induce anti-hantavirus antibody to measure Hantaan virus sample particle institute's inductive antibody and natural Hantaan virus antigen and the antigenic reaction of reorganization Hantaan virus in animal body by immunofluorescence (IFA) and enzyme linked immunosorbent assay (ELISA).1, used the antigen sheet of the insect cell preparation of the Vero-E6 cell that infects by natural Hantaan virus, the insect cell of expressing Hantaan virus glycoprotein, expression Hantaan virus nucleoprotein among the IFA:IFA.Rabbit immune serum serial dilution with preparation described in the embodiment 11 is added drop-wise on the different antigen sheets, hatches 30 minutes for 37 ℃, wash 3 times with PBS, dry up the back and drip fluorescein-labeled second antibody, hatched 30 minutes for 37 ℃, wash 3 times with PBS, dry up under the rearmounted fluorescent microscope and pass through observation.The virus-like particle inductive immune serum and the Vero-E6 cell of natural Hantaan virus infection, the insect cell of expression Hantaan virus glycoprotein, the antigen sheet that the insect cell of expression Hantaan virus nucleoprotein prepares that are formed jointly by Hantaan virus glycoprotein and nucleoprotein are positive.The Vero-E6 cell that virus-like particle inductive immune serum that is formed separately by Hantaan virus glycoprotein and natural Hantaan virus infect, the antigen sheet of expressing the insect cell of Hantaan virus glycoprotein are positive and express the reaction that is negative of antigen sheet that the insect cell of Hantaan virus nucleoprotein prepares.2, ELISA: use indirect method ELISA to detect, insect cell with the Vero-E6 cell of natural Hantaan virus infection, the insect cell of expressing Hantaan virus glycoprotein, expression Hantaan virus nucleoprotein carries out the centrifugal preparation envelope antigen of ultrasonication, after the titration of envelope antigen process, coated elisa plate.Rabbit immune serum serial dilution with preparation described in the embodiment 11, be added to and be coated with in the antigenic enzyme plate of different Hantaan virus, every hole 100 μ l were hatched 60 minutes for 37 ℃, washed plate 6 times with PBS-Tween20, add corresponding enzyme mark second antibody respectively, hatched 60 minutes for 37 ℃, wash plate 6 times, add the substrate colour developing with PBS-Tween20, measure the 0D value in every hole with microplate reader, determine antibody titers.The virus-like particle inductive immune serum and the Vero-E6 cell of natural Hantaan virus infection, the insect cell of expression Hantaan virus glycoprotein, the antigen that the insect cell of expression Hantaan virus nucleoprotein prepares that are formed jointly by Hantaan virus glycoprotein and nucleoprotein are positive.The Vero-E6 cell that virus-like particle inductive immune serum that is formed separately by Hantaan virus glycoprotein and natural Hantaan virus infect, the antigen of expressing the insect cell of Hantaan virus glycoprotein are positive and express the reaction that is negative of antigen that the insect cell of Hantaan virus nucleoprotein prepares.
Embodiment 13 Hantaan virus sample particles induce the anti-hantavirus neutralizing antibody by PRNT measure Hantaan virus sample particle in animal body institute's inductive at the neutralizing antibody of Hantaan virus.Cultivate the Vero-E6 cell in 24 hole tissue culturing plates, cell grows up to behind the individual layer stand-by.To put 56 ℃ of deactivations in 30 minutes with the rabbit immune serum of Hantaan virus sample granules preparation, use then aseptic cell culture fluid with rabbit anteserum since 1: 80 2 times of serial dilution, get each extent of dilution serum, contain the viral liquid balanced mix of 20-30PFU Hantaan virus prototype-strain-76118 strain with per 100 μ l, put 4 ℃ of neutralizations and spend the night.Long 24 orifice plates that fine and close individual layer Vero-E6 cell is arranged of inoculation next day, every hole adds 200 μ l, and the parallel work of each serum dilution 2 holes are set up positive serum contrast and virus control simultaneously.Every hole, absorption back adds the agarose that dissolves that 1ml contains cell culture fluid, treats after agarose solidifies Tissue Culture Plate to be put the carbonic acid gas incubator, at 37 ℃, 5%CO
2Environment was cultivated 7-9 days down.Add the agarose that contains toluylene red, treat after agarose solidifies Tissue Culture Plate to be put the carbonic acid gas incubator, at 37 ℃, 5%CO
2Environment is cultivated down, observes the plaque that Hantaan virus forms.Be judged as the positive more than 50% to reduce, determine the NAT of immune serum than virus control plaque number.NAT with two rabbit of the common virus-like particle immunity that forms of Hantaan virus glycoprotein and nucleoprotein was respectively 1: 640 and 1: 640, was respectively 1: 640 and 1: 320 with the NATs of two rabbit of the virus-like particle immunity of Hantaan virus glycoprotein formation.
Description of drawings 1, Fig. 1 show by Hantaan virus glycoprotein form the Hantaan virus sample of justacrine in the culture supernatant in the CHO engineering cells
Particle, the wolframic acid negative staining is used in the purified back of Hantaan virus sample particle, amplifies 31000 times form under electron microscope.2, Fig. 2 demonstration forms justacrine in culture supernatant jointly by Hantaan virus glycoprotein and nucleoprotein in the CHO engineering cell
Hantaan virus sample particle, the wolframic acid negative staining is used in the purified back of Hantaan virus sample particle, amplifies 31000 under electron microscope
Form doubly.3, Fig. 3 is that Hantaan virus sample particle behind the purifying is after SDS-PAGE separates, with examining the painted result of Ma Shi light blue.The 1st swimming
The road sample is the common virus-like particle that forms of Hantaan virus glycoprotein and nucleoprotein, show behind the electrophoresis glycoprotein g1, G2 and
The nucleoprotein band.The 2nd swimming lane sample is the virus-like particle that Hantaan virus glycoprotein forms separately, shows sugared egg behind the electrophoresis
White G1 and G2 band.The 3rd swimming lane is the molecular weight of albumen mark.4, Fig. 4 is the Western blot result of the smooth virus-like particle of the purifying Later Han Dynasty, the 1st swimming lane sample be Hantaan virus glycoprotein and
The common virus-like particle that forms of nucleoprotein, can with the monoclonal antibody reactive of anti-hantavirus nucleoprotein, show positive
Band.The 2nd swimming lane sample is the virus-like particle that Hantaan virus glycoprotein forms separately, can examine egg with anti-hantavirus
Behind the white monoclonal antibody reactive, positive band does not appear.5, Fig. 5 is the virus-like particle that forms separately of Hantaan virus glycoprotein and the reaction of different antibodies, this particle can and polyclone
Serum and anti-hantavirus glycoprotein monoclonal antibody reactive, can not with anti-hantavirus nucleoprotein monoclonal antibody reactive.6, Fig. 6 is the common virus-like particle that forms of Hantaan virus glycoprotein and nucleoprotein and the reaction of different antibodies, and this particle can
With anti-hantavirus polyclonal serum, anti-hantavirus glycoprotein monoclonal antibody and anti-hantavirus nucleoprotein monoclonal anti
Precursor reactant.
Claims (11)
1, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: every kind of engineering cell system is integrated with M segment and the S segment genome or the single M segment genome of above-mentioned various Hantaan virus of HTN (HTN), soul type (SEO), palm fibre back of the body flounder type (PUU), four serotype hantaan types of Du's Bulova type (DOB) virus respectively.
2, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: engineering cell system is divided into two classes, but a class is expressed the Hantaan virus glycoprotein g1 of various hantaan type virus M segment genome coding and the Hantaan virus nucleoprotein of G2 and S segment genome coding for jointly stabilizing; Another kind of Hantaan virus glycoprotein g1 and the G2 that expresses various hantaan type virus M segment genome coding for single stable.
3, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: common continual and steady the expression or the single continual and steady expression of Hantaan virus glycoprotein in mammalian cell in mammalian cell by Hantaan virus glycoprotein and nucleoprotein, can in cell, form the virus-like particle justacrine to the extracellular, thus in cells and supernatant sustainable results Hantaan virus sample particle.
4, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: the Hantaan virus sample particle that any type integrator gene described in this group clone Sustainable Production claim (1) and (2) in the four type Hantaan virus is determined, comprising the virus-like particle of Hantaan virus glycoprotein and the formation of nucleoprotein co expression and the virus-like particle of the single expression formation of Hantaan virus glycoprotein.
5, claim (1-4) is produced Hantaan virus sample particulate Mammals engineering cell system for described one group, it is characterized by: this cell is Chinese hamster ovary cell (Chinese hamster ovary celI) and African green monkey kidney cell (Vero cell), also can be any possible cells of mamma animals that is used to produce.
6, according to claim (4), produce the Hantaan virus sample particle that different Hantaan virus types that Hantaan virus sample particulate Mammals engineering cell system produced and different genes are formed, can use in external independent or any compatible combination for one group according to the needs of practical application from now on.
7, produce Hantaan virus sample particulate Mammals engineering cell system for one group; it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed; have with the glycoprotein of natural Hantaan virus and nucleoproteide like immunogenicity; no matter be that single type reorganization Hantaan virus sample particle or different type Hantaan virus sample particle compatibility use, all can inducing producing specificity neutralizing antibody and other corresponding immune responses in animal or human's body.
8, produce Hantaan virus sample particulate Mammals engineering cell system for one group, it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed, have with the glycoprotein of natural Hantaan virus and nucleoproteide like antigenic characteristic, can induce the polyclone of generation and monoclonal antibody generation specificity to combine with natural Hantaan virus structural protein.
9, according to claim (1-8); one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system; it is characterized in that: the Hantaan virus sample particle that different Hantaan virus types that this group clone is produced and different genes are formed; can be used to prepare the Hantaan virus vaccine; can prepare the Hantaan virus vaccine with single type Hantaan virus sample particle or different type Hantaan virus sample particulate various combination; after these vaccines use in the crowd, can prevent other Hantaan virus of different shaped to infect.
9, according to claim (1-8); one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system is characterized in that: one group of Hantaan virus reorganization nucleoprotein, recombinant glycoprotein, virus-like particle or their combination of expressing with mammal cell line constitutes Hantaan virus diagnostic kit and Hantaan virus classification diagnosis test kit as antigen component.
10, according to claim (1-8), one group of purposes of producing Hantaan virus sample particulate Mammals engineering cell system, it is characterized in that: by various molecular biology methods, the gene that has therapeutic action in Hantaan virus sample granule interior packing, these genes can be RNA or DNA, by Hantaan virus sample particle with these transgenosis in body, as the transfer vector of gene therapy.
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| CN02104382A CN1442479A (en) | 2002-03-05 | 2002-03-05 | Mammalian engineering cell line for production of hantan virus granules |
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| CN02104382A CN1442479A (en) | 2002-03-05 | 2002-03-05 | Mammalian engineering cell line for production of hantan virus granules |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812478B (en) * | 2009-12-29 | 2012-07-25 | 中国人民解放军第四军医大学 | Adenovirus high expression vector for improving expression of hantavirus fusion protein G1S0.7 |
| CN104946605A (en) * | 2015-07-06 | 2015-09-30 | 中国人民解放军第四军医大学 | Hantavirus sample particle with CD40L and preparation method and application of hantavirus sample particle |
| CN104974990A (en) * | 2015-07-06 | 2015-10-14 | 中国人民解放军第四军医大学 | Hantaan virus-like particle containing GM-CSF as well as preparation method and application of hantaan virus-like particle |
-
2002
- 2002-03-05 CN CN02104382A patent/CN1442479A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812478B (en) * | 2009-12-29 | 2012-07-25 | 中国人民解放军第四军医大学 | Adenovirus high expression vector for improving expression of hantavirus fusion protein G1S0.7 |
| CN104946605A (en) * | 2015-07-06 | 2015-09-30 | 中国人民解放军第四军医大学 | Hantavirus sample particle with CD40L and preparation method and application of hantavirus sample particle |
| CN104974990A (en) * | 2015-07-06 | 2015-10-14 | 中国人民解放军第四军医大学 | Hantaan virus-like particle containing GM-CSF as well as preparation method and application of hantaan virus-like particle |
| CN104946605B (en) * | 2015-07-06 | 2019-04-23 | 中国人民解放军第四军医大学 | Hantaan virus sample particle comprising CD40L and the preparation method and application thereof |
| CN104974990B (en) * | 2015-07-06 | 2019-05-21 | 中国人民解放军第四军医大学 | Hantaan virus sample particle comprising GM-CSF and the preparation method and application thereof |
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