CN105349498B - A kind of method that optimization prepares inactivated vaccine and/or attenuated live vaccine - Google Patents

Abstract

The present invention provides a kind of method that optimization prepares inactivated vaccine and/or attenuated live vaccine, press down cancer candidate gene 2 by being overexpressed glioma in host cell, optimization is used to prepare copying surroundings in the host cell of inactivated vaccine and/or attenuated live vaccine, improve 4 section viroids (including coronaviridae, Paramyxoviridae, orthomyxoviridae family and herpetoviridae) 1-3 lg unit of potency, the present invention provides new strategy and feasible foundation to optimize the virus titer of whole virus vaccine, field of biomedicine is with important application prospects and innovative significance.

Description

A kind of method that optimization prepares inactivated vaccine and/or attenuated live vaccine

Technical field

The present invention relates to whole virus vaccine preparation fields, prepare inactivated vaccine specifically, being related to a kind of optimization and/or subtract The method of virus live vaccine.

Background technique

Whole virus vaccine (including Attenuate vaccine and inactivated vaccine) still occupies sizable specific gravity in modern vaccination application.Entirely Viral vaccine is otherwise known as conventional vaccine, including inactivated vaccine and attenuated live vaccine, and wherein attenuated live vaccine is using artificial fixed Vaccine made of the microorganism of virulence height decrease or substantially nontoxic work is filtered out to the method for variation, or from nature.Subtract After virus live vaccine inoculation, there is certain growth and breeding ability in body, immune effect is strong and lasting, general to be only inoculated with one Secondary, immunocompetence can be kept throughout one's life, in addition to stimulation body generates cellular immunity and humoral immunity, can still generate local immunity guarantor Shield.According to this standard, best vaccine is undoubtedly exactly living, attenuation pathogen, however dissipating poison and threatening is that it always can not The problem of avoidance.Inactivated vaccine is to select the strong pathogenic microorganism of immunogenicity through cultivating, with either physically or chemically being gone out After work, then purified it is made.Inactivated vaccine has lost the appeal to body, but still keeps its immunogenicity, can stimulate machine Body generates corresponding immunity, resists the infection of street strain.Inactivated vaccine often need to be repeatedly inoculated with, and inoculation is not generated once to have and be protected Being immunized for shield effect, only " initializes " immune system.Non- whole virus vaccine can reduce the wind of virulence reverse to the maximum extent Danger, wherein subunit vaccine is only a small amount of antigen portion in the how species specific antigenic determinant that macromolecular antigen carries Position plays an important role to protective immune response.Native protein is separated by protein-hydrolysis process such as chemical breakdowns, is mentioned Viral special area is taken, is filtered out with vaccine made of immunocompetent segment.Subunit vaccine can be eliminated many unrelated anti- Original induce antibody, thus reduce vaccine side reaction and vaccine caused by related disease.Recombinant vaccine, which refers to, utilizes DNA Restructuring biotechnology will can induce the natural or artificial synthesized inhereditary material of immune response in pathogen coat protein In orientation insertion bacterium, yeast or mammalian cell, be allowed to give full expression to, it is purified after prepared by vaccine.But this two The shortcoming of class vaccine is that immunogenicity is lower, and not comprehensively, the immune effect that could have been generated need to be shared with adjuvant, therefore Optimal protecting effect is often not achieved.

The problem that whole virus vaccine preparation field faces at present is that many production of vaccine are not high with strain potency, this is directly Affect the effect of vaccine.By seedling poison under different preservation conditions, discovery potency titre is presented one for domestic and international some seminars The variation for rule of establishing rules, and finally determine suitable preservation condition.There are also some seminars to improve vaccine effectiveness using immunologic adjuvant, Such as inorganic adjuvant (aluminium hydroxide), organic adjuvant (bacterial product etc.), synthetic adjuvant (levamisol etc.), finish (Fei Shi assistant Agent etc.) and the immunosuppressor such as ciclosporin A, rapamycin.However, the adjuvant types that country formally examines are very limited, It does not advocate for human body, these adjuvants are mainly used for zoopery, and adjuvant disease can often occur after animal multiple injection, therefore help The requirement of agent is very careful, is also extremely restricted.In addition, other seminars carry out strain phenotype with strain to production Or science of heredity transformation, make it more suitable for the exploitation of Attenuate vaccine or inactivated vaccine.There are also researchers to attempt different cell lines, will such as paste Parietal cell changes into suspension cell, to promote the potency titre of cell Proliferation virus by the cell density for increasing unit volume. Above scheme improves the potency titre of vaccine virus to a certain extent, but there is no thin by understanding the host of virus replication in depth Born of the same parents' factor finds and develops the intracellular resource for being conducive to virus replication.

Summary of the invention

The object of the present invention is to provide a kind of methods that optimization prepares inactivated vaccine and/or attenuated live vaccine.

In order to achieve the object of the present invention, the present invention provides glioma suppression (the Glioma tumor of cancer candidate gene 2 Suppressor candidate region gene 2, GLTSCR2) coding albumen P60 preparing inactivated vaccine and/or attenuation Application in live vaccine, the glioma suppression cancer candidate gene 2 encode the amino acid sequence of albumen as shown in Seq ID No.1, Or the sequence is through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.

P60 albumen is a kind of ribosomal protein P60 for being positioned at entoblast, and 4 class virus titer 1-3 can be improved Lg unit, including it is coronavirus (such as infectious bronchitis virus), paramyxovirus (such as canine distemper and newcastle disease virus), just viscous Viral (such as influenza virus) and herpesviral (such as herpes simplex virus and Marek's disease poison).Molecular mass is the P60 of 60kDa Albumen and Cycle Regulation, DNA damage response and relationship with apoptosis are very close.It is reported that in DNA damage, Nucleolin P60 can be positioned to cell caryoplasm, and in conjunction with important tumor suppressor P53, inhibit thin while stablizing P53 Intracellular growth；On the other hand, in order to cope with kernel stress, P60 albumen can block ribosomal protein in conjunction with MDM2, to promote The degradation of P53 ubiquitination.In addition, PI3K-Akt/PKB signal pathway cell survival and in terms of all play important work With PTEN is the main brake albumen of the access, but PTEN can degrade rapidly after P60 gene knockout, and lead to tumour.Cause , in oncology, P60 possesses " tumor suppressor " and " roadblock for inhibiting cancer " dual identity for this.The unique biochemistry of P60 Characteristic and sophisticated functions are allowed to rapidly become scientific research hot spot, but in field of virology, rarely seen document report.

Present invention firstly discovers that P60 albumen can be functioned by several cellular pathways, such as regulation innate immune system, Cell defense system, protein-synthesizing system.

The present invention provides a kind of method for preparing inactivated vaccine and/or attenuated live vaccine of optimization, the mistake in host cell It expresses glioma and presses down cancer candidate gene 2, and be inoculated with host cell, training with inactivated vaccine and/or attenuated live vaccine production strain Cell is supported, virus liquid is harvested.

The inactivated vaccine and/or attenuated live vaccine include but is not limited to coronaviridae (such as infective bronchitis disease Poison), Paramyxoviridae (such as canine distemper and newcastle disease virus), orthomyxoviridae family (such as influenza virus) and herpetoviridae be (as single Pure herpesviral and Marek's disease poison) etc. inactivated vaccine and/or attenuated live vaccine.

Method above-mentioned, when the convergence degree of host cell reaches 80~90%, by 1~10 TCID₅₀Inoculation inactivation epidemic disease Strain is used in seedling and/or attenuated live vaccine production.

The cell culture mode used in the present invention is adhere-wall culture or the culture that suspends.

The present invention also provides a kind of host cells of overexpression glioma suppression cancer candidate gene 2.

The present invention also provides above-mentioned host cells to prepare the application in inactivated vaccine and/or attenuated live vaccine.

The present invention by host cell be overexpressed coding P60 albumen gene, optimization be used to prepare inactivated vaccine and/ Or copying surroundings in the host cell of attenuated live vaccine, it is (including coronaviridae, Paramyxoviridae, just viscous to improve 4 section viroids Viraceae and herpetoviridae) 1-3 lg unit of potency, the present invention for optimize whole virus vaccine virus titer provide new plan Slightly with feasible foundation, field of biomedicine is with important application prospects and innovative significance.

Detailed description of the invention

Fig. 1 is characteristic and function of the p60 in virus infection host cell；Wherein, the albumen p60 of A1, two kinds of cells exist Cellular localization when being uninfected by, it is seen that be located in nucleus；A2, after human herpes simplex vicus -1 (HSV-1) infection, it is seen that P60 is migrated to cytoplasm；A3, after newcastle disease virus (NDV) infection, it is seen that p60 is migrated to cytoplasm；B, HSV-1 infection (10TCID₅₀) after Hep-2 different time, collect cell sample and carry out nucleus-matter separation, then carried out using each antibody Western-blot analysis, it is seen that the positioning variation tendency for going out core after core occur first entering in p60 and its chaperone PTEN；C, DNA synthetic inhibitor PMEG (+) is added in virus infection simultaneously, after different time (experiment condition and A are identical as B), it is seen that PMEG (10 μM) will not influence the total expression of p60, but can significantly affect cellular localization, thus it is speculated that it is direct with virus replication that p60 goes out core It is related；D is overexpressed GFP-N1 or GFP-p60 (10 μ g) after PTEN knockout 96h (800ng), and 40h postoperative infection HSV-1 is different Time, it is seen that the knockout of PTEN acts on the enhancement of virus replication without inhibiting to be overexpressed p60, therefore p60 function and PTEN is not fully related.

Fig. 2 is the influence for being overexpressed or knocking out 2 kinds of virus replications of p60 gene pairs in embodiment；Wherein, A1, from left to right It is followed successively by, Hep-2 cell is overexpressed control GFP-N1 plasmid postoperative infection NDV, is overexpressed GFP-p60 plasmid postoperative infection NDV, only feels It contaminates NDV, Hep-2 cell and knocks out (Neg) infection NDV, knockout control p60 gene postoperative infection NDV after control scramble gene, institute With 10 μ g of plasmid, being overexpressed the time is 40 hours (A2 is same)；Knockout siRNA dosage is 800ng, and the time is 96 hours (A2 is same)； Infection time is 48 hours, 10 TCID₅₀(A2 is same) then surveys virus titer using cytopathy political reform；A2, from left to right successively For, in addition to p60 sample is repeated 1 times (label 2-3), other sample treatments and A1 are same, virus protein N P antibody used, and 1: 1000 dilutions.It can be seen that p60 knockout is unfavorable for NDV duplication, and virus replication can then be optimized by being overexpressed；B1 is followed successively by from left to right, Hep-2 cell be overexpressed control GFP-N1 plasmid postoperative infection HSV-1, be overexpressed GFP-p60 plasmid postoperative infection, only infection HSV-1, Cell knocks out control scramble gene postoperative infection, knocks out control p60 gene postoperative infection, and 10 μ g of plasmid used is overexpressed the time It is 40 hours (B2 is same)；Knockout siRNA is 800ng, and the time is 96 hours (figure B2 is same)；Infection time is 48 hours, 10 TCID₅₀(B2 is same) then surveys virus titer using cytopathy political reform；B2 is followed successively by from left to right, cell infection HSV-1, crosses table Up to control GFP-N1 plasmid postoperative infection, it is overexpressed GFP-p60 plasmid postoperative infection, cell knockout control scramble, knockout control Scramble postoperative infection knocks out control p60, knocks out control p60 postoperative infection HSV-1.Virus protein ICP8 used and ICP0 antibody, It is 1:1000 dilution；It can be seen that p60 knockout is unfavorable for HSV-1 duplication, and virus replication can then be optimized by being overexpressed.It can be seen that obvious poor It is different, p < 0.01 * *.

Fig. 3 is the influence for knocking out 4 kinds of virus replications of p60 gene pairs；Wherein, primary fibroblast CEF knocks out control Scramble gene (800ng), or control p60 gene (800ng) is knocked out, 96 hours.Then A1 infects canine distemper virus (CDV) (10 TCID₅₀) different time, the transcriptional level of RT-PCR method survey viral gene N, it is seen that difference, p < 0.05 *.Or it strikes Except A2 is carried out after 96 hours, indirect immunofluorescence is observed in infection after CDV virus-4 8 hours, calculates median infective dose TCID₅₀, it is seen that Notable difference, p < 0.01 * *.

B, CEF cell knock out control scramble gene (800ng), or knock out control p60 gene (800ng), and 96 hours. Then B1 infects marek virus (MDV) (10 TCID₅₀) different time, the transcription water of RT-PCR method survey viral gene Meq It is flat, it is seen that difference, p < 0.05 *.Or B2 is carried out after knocking out 96 hours, infection observes cytopathy after MDV virus-4 8 hours, calculates Median infective dose TCID₅₀, it is seen that difference, p < 0.05 *.

C, CEF cell knock out control scramble gene (800ng), or knock out control p60 gene (800ng), and 96 hours. Then C1 infects infectious bronchitis virus (IBV) (10 TCID₅₀) different time, then viral base is surveyed with RT-PCR method Because of the transcriptional level of N, it is seen that difference, p < 0.05 *.Or C2 is carried out after knocking out 96 hours, it is collected after infection IBV virus-4 8 hours thin Lesion is observed in born of the same parents, cracking for postoperative infection 9 days after instar chicken embryo, calculate chicken embryo median infective dose EID₅₀, it is seen that difference, p < 0.05 *.

D, Vero cell knock out control scramble gene (800ng), or knock out control p60 gene (800ng), and 96 hours Postoperative infection avian influenza virus (AIV) virus, observes cytopathy after 48 hours, calculate median infective dose TCID₅₀, it is seen that difference, * p<0.05。

Fig. 4 is the influence for being overexpressed p60 to 4 kinds of virus replications；Wherein primary fibroblast CEF Transfection of GFP-N1 or GFP-p60 plasmid, each 10 μ g；A1, i.e. different time postoperative infection CDV, 10 TCID are carried out after 40 hours₅₀, then use RT- The transcriptional level of PCR method survey viral gene N, it is seen that after infection 6 hours, significant difference, p < 0.01 * *；Or A2, virus infection 48 Indirect immunofluorescence is observed after hour, calculates median infective dose TCID₅₀, it is seen that difference, p < 0.05 *.

CEF cell transfecting GFP-N1 or GFP-p60 plasmid, each 10 μ g；B1, i.e. different time postoperative infection are carried out after 40 hours MDV, 10 TCID₅₀, the transcriptional level of viral gene Meq is then surveyed using RT-PCR method, it is seen that after infection 6 hours, difference is aobvious It writes, p < 0.01 * *；Or B2, virus infection observe cytopathy after 48 hours, calculate median infective dose TCID₅₀, it is seen that difference, * p <0.05。

CEF cell transfecting GFP-N1 or GFP-p60 plasmid, each 10 μ g；C1, i.e. different time postoperative infection are carried out after 40 hours IBV, 10 TCID₅₀, the transcriptional level of viral gene N is then surveyed using RT-PCR method, it is seen that after infection 6 hours, difference is aobvious It writes, p < 0.01 * *；Or C2, virus infection collected cell after 48 hours, infected instar chicken embryo on the 9th, calculated chicken embryo median infective dose EID₅₀, it is seen that difference, p < 0.05 *.

D, Vero cell transfecting GFP-N1 or GFP-p60 plasmid, each 10 μ g, 40 hours postoperative infection AIV virus, 10 TCID₅₀, cytopathy is observed after 48 hours, calculates median infective dose TCID₅₀, it is seen that difference, p < 0.05 *.

In A1, B1, C1, with 2^-△△CTMethod calculates the difference of virus mRNA expression between different disposal group.Use GraphPad Prism5 draws.The N1 processing of each time point is all standardized as 1.

Fig. 5 is the mechanism of action that p60 can optimize virus replication；Wherein, A is followed successively by from left to right, Hep-2 cell transfecting SiRNA-negtive 200ng (40 μ l), transfection siRNA-negtive800ng (160 μ l), siRNA-p60200ng (40 μ l), SiRNA-p60400ng (80 μ l), siRNA-p60800ng (160 μ l) are transfected, knocking out the time is 96 hours, is used before sample harvest Poly (I:C) is stimulated 6 hours, and then RT-PCR method surveys IFN-β transcriptional level, it is seen that significant difference, p < 0.01 * *；B, sequentially with Method is identical as A, and RT-PCR method surveys MAVS transcriptional level, significant difference, p < 0.01；C, sequentially and method is identical as A, RT-PCR Method surveys NF- к B transcriptional level, significant difference, p < 0.01；D, sequentially and method is identical as A, and RT-PCR method surveys MDA5 transcriptional level. Transfection knock out p60 with compare between indifference it is significant, although certain concentration dependent, p < 0.05 is presented in p60；E, sequentially with side Method is identical as A, RT-PCR method survey RIG-1 transcriptional level, transfection knock out p60 with compare between indifference it is significant, although p60 presentation Certain concentration dependent, p < 0.05；F is followed successively by from left to right, is uninfected by Hep-2 cell, is overexpressed control GFP-N1 matter Grain is overexpressed GFP-p60 plasmid, is overexpressed control GFP-N1 plasmid postoperative infection HSV-1, is overexpressed GFP-p60 plasmid postoperative infection, Each protein expression level is then surveyed using western-blot method, 10 μ g of plasmid used, being overexpressed the time is 40 hours；Infection Time is 48 hours, 10 TCID₅₀(E is same), as a result as it can be seen that the unfavorable albumen of p60 synthesizes (phosphorylation eIF2 α), but virus can rob It holds and weakens this process.

Fig. 6 show pEGFP-N1 plasmid map in the embodiment of the present invention 2.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.

In following embodiment:

NDV strain is F48E9；IBV strain is H52；CDV is standard strain Snyder Hill；HSV-1 be standard poison F, MDV strain is RB1B, and AIV is H9N2 separation poison.

The cell median infective dose and chicken embryo median infective dose of several viruses of embodiment 1

It takes 9-11 age in days SPF chicken embryo to prepare chicken embryo fibroblasts (CEF), CDV, MDV and IBV is inoculated on CEF and trained 48h is supported, cell and culture supernatant multigelation 3 times is harvested, is inoculated on CEF cell again and continues to cultivate, be so passaged to 3 Generation.By 10 doubling dilution of virus liquid, 96 orifice plate of inoculating cell or chicken embryo.CDV surveys cell median infective dose (TCID₅₀), MDV is with carefully TCID is surveyed in the formation of born of the same parents' lesion pox spots₅₀, IBV chicken embryo median infective dose EID₅₀, determine 3 kinds of viral virulence.It the results are shown in Table 1- Table 3.

The influence (indirect immunofluorescence) of 1 CDV cell median infective dose of table

It is calculated by Reed and Muench formula: distance proportion=(the lesion rate -50 higher than 50%)/(disease higher than 50% Variability-is lower than 50% lesion rate).TCID is calculated₅₀=10^4.38/ 0.1ml, as TCID₅₀=10^5.38/ml。

The influence (cytopathy political reform) of 2 MDV cell median infective dose of table

TCID is calculated₅₀=10^3.70/ 0.1ml, as TCID₅₀=10^4.70/ml。

The influence (chicken embryo short and smallization method) of 3 IBV chicken embryo median infective dose of table

EID is calculated₅₀=10^5.5/ 0.1ml, as EID₅₀=10^6.5/ml。

Similarly calculate the TCID of NDV₅₀=2 × 10⁹The TCID of/ml, HSV-1₅₀=10^8.1The TCID of/ml, AIV₅₀= 10^5.4/ml。

Embodiment 2 knocks out the influence (Western blot, lesion method and RT-PCR method) with overexpression P60 to virus virulence

1, the preparation of cell

Pass on Hep-2 and Vero cell: growth-promoting media contains the DMEM culture solution of 5% fetal calf serum and 100U mycillin, The DMEM culture solution of 2% fetal calf serum of maintaining liquid and 100U mycillin.

Primary chick embryo fibroblast (CEF) cell: instar chicken embryo is purchased from Beijing Cimmeria company within 9-10 days, and catalog number is " SPF chicken embryo ".After 9-10 days instar chicken embryos are taken out carefully will end to end, bone and adipose tissue reject, remaining part is washed with PBS It is cut into small pieces as far as possible after 3 times, 0.25% pancreatin is added to digest 30 minutes, 70ml culture solution is added after washing 3 times in PBS, and (growth-promoting media contains There is a DMEM culture solution of 5% fetal calf serum and 100U mycillin, 2% fetal calf serum of maintaining liquid and 100U mycillin DMEM culture solution), fibroblast is flushed out energetically, and culture solution containing cell crosses three layers of gauze, with 5ml/ bottles or 200 μ l/ The packing of 24 orifice plates, 37 DEG C after cell culture 24 hours (coverage rate up to 80% or more) can carry out next step experiment.

HSV and NDV infects Hep-2 cell, and AIV infects vero cell, and CDV, MDV and IBV infect CEF cell.In addition, thin Born of the same parents' culture solution is purchased from Beijing Mai Chen company, catalog number CM15019.

2, viral proliferation

The cytotoxic proliferation of HSV, NDV, AIV and CDV: according to the amount virus inoculation stoste of cell bottle culture solution 1/10, to Cytopathy (such as NDV forms big plasomidum) is viral using multigelation method harvest up to 75% (generally 48 hours), i.e., will be thin Born of the same parents bottle be put into -20 DEG C freeze 2 hours after place room temperature to partial melting, yawing cell bottle makes attached cell take off wall at this time, again It is put into -20 DEG C to freeze, 3 times repeatedly, releases virus from cell, frozen respectively at -20 DEG C and -70 DEG C.

The proliferation of the MDV virus of cell dependent antibody: its be proliferated with save the difference is that, do not use multigelation method receive Virus is obtained, i.e., the virus of Liquid nitrogen storage is placed and is slowly melted on ice, be then inoculated with disease according to the amount of cell bottle culture solution 1/10 Toxogen liquid, when cytopathy is up to 75% (generally 48~72 hours), the digestion of 0.25% pancreatin makes attached cell take off wall, again It is put into liquid nitrogen cryopreservation.

The proliferation of IBV embryo toxicity: 9-10 days instar chicken embryos are through the every embryonic breeding kind 10 of allantoic cavity⁴A TCID₅₀Virus stock solution used, inoculation Chicken embryo collects allantoic fluid after 140 hours, -20 DEG C or -70 DEG C freeze preservation, and IBV infection chicken embryo is not dead, but the obvious shape of chicken embryo Body is smaller (dwarf embryo).

3, the measuring method of virus titer titre

Cytopathy (cytopathic effect, CPE) is cytopathic effect, refers to viruses into tissues culture cell The cell degeneration generated after infecting can carry out Viral Quantification using such pathological effect.It is common that virus infection forms cytopathy Plasomidum (maxicell that i.e. multiple cell aggregations are formed together multicore) and two kinds of plaque (cell detachment formation plaque).Lesion is melted Conjunction rate is the ratio that sick cell accounts for all cells.

Toxicity test: obtaining cell toxicant suspension for cell toxicant doubling dilution, with 96 orifice plate culture CEF cells to single layer, to 50 hole μ L/ (including HSV, NDV, MDV and AIV) of cell toxicant suspension is added in each hole, 12 dilutions and 8 repetitions are done, in 37 Cytopathy is observed after placing 48 hours in DEG C carbon dioxide incubator, and records lesion fusion rate (lesion fusion rate i.e. lesion Cell accounts for the ratio of all cells), it is repeated 3 times.Indirect immunofluorescence: the CFE cell culture medium that malicious CDV is cultivated 5 days will be attacked and inhaled It abandons, after washing 3 times with PBS, after adding 4% formaldehyde fixed, is incubated for 60min with CDV mouse monoclonal antibody, is washed 3 times, added with PBS The secondary antibody for entering the mountain sheep anti mouse of Rhodamine Red-x label is incubated for 40min, is washed 3 times with PBS.In immunofluorescence microscopy Under 568nm wavelength, by observing and recording virus fluorescence, to carry out toxicity test.

TCID₅₀Calculation method: virus is serially diluted, is laterally inoculated with cell monolayer plate by 96 orifice plate cell monolayers of preparation, Every dilution repeats 3 holes, observes cytopathy daily, and viral dilution of the record higher than 50 and lower than 50% lesion hole calculates Than away from acquisition TCID₅₀As a result.Calculation formula are as follows: (the lesion rate -50% higher than 50%)/(higher than 50% lesion rate-it is less than 50% lesion rate)=than away from；Than just obtaining index away from being added with the index of the dilution of the virus close to 50% lesion rate. For example, colorimetric calculates or the TCID of micro- sem observation virus₅₀10^-7~10^-8Between, then, index -8 with than obtaining away from being added The new index obtained, as TCID₅₀Index.

IBV toxicity test: 9-10 days instar chicken embryos are through allantoic cavity virus inoculation solution, every 1 μ L of egg inoculation (1 μ L virus The virus quantity contained in solution is 10⁴A TCID₅₀Unit) viral solution, inoculated into chick embryo collects allantoic fluid after 48-72 hours, see Examine chicken embryo lesion.As a result, the pathological condition that chicken embryo body becomes smaller is presented in chicken embryo, (body trunk portion length is about normal control chicken The 2/3 of idiosome product), it is contemplated that hereafter the situation of virus often high titre infection in actual production is tested all using this viral dilution Degree.

4, real-time quantitative RT-PCR method and the primer

The design of primer: according to the viral protein gene sequence delivered in GenBank, such as CDV-N albumen, MDV-meq Albumen, IBV-N protein sequence carry out nucleotide homology analysis using molecular biology software DNAStar, select virus replication Target sequence of the relevant gene conserved sequence as PCR amplification.Fluorescence PCR primer design principle is followed, it is soft with Primer 5.0 Part separately designs primer (table 4).

Real-time quantitative PCR: siRNAP60 and siNeg are transfected to CEF 96h, during which by N1, P60 plasmid transfection to CEF After upper 40h, virus inoculation extracts total serum IgE according to Trizol lysate specification 3 after connecing poison, 6,12,24,48h receipts cell. Specific downstream primer is used to carry out the synthesis of cDNA as reverse transcription primer.Real-time quantitative is carried out with SYBR Green I dye method PCR amplification.20 μ L overall reaction systems: 10 μ 2 × SYBR of L Green I PCR mix, each 0.4 μ L of upstream and downstream primer (10 μM), Moisturizing is to 20 μ L.Real-Time PCR System (ViiA7) sets response procedures: 95 DEG C of initial denaturation 5min, is denaturalized 95 DEG C 10s, 60 DEG C of annealing and extension 45s, set 40 circulations, finally carry out melting curve analysis.Take 2^-ΔΔtMethod, to target gene Relative quantification is carried out, β-actin is selected as reference gene.

4 primer sequence information of table

5, gene knockout step

Cell transfecting: taking the SPF chicken embryo of 9-11 age in days to prepare CEF or passage cell Hep-2 and Vero, cell density extremely After 50%, siRNAP60 and siNeg (each 800ng) are distinguished according to (invitrogen) specification of lipofectamine 2000 Transfect 96h.Target P60 gene specific small RNA fragments Sip60, and the Neg without selectively targeted any cell protein sequence (or scramble) is purchased from sub- novacine object limited liability company (Abnova).

Virus inoculation: Transfected cells distinguish virus infection, the equal 10TCID of every kind of virus₅₀, cultivate different time.

6, it is overexpressed

Cell transfecting: CEF, Hep-2 or Vero cell reach 80% to cell density, according to lipofectamine 2000 specifications transfect eukaryon expression plasmid pEGFP-P60 and its carrier (pEGFP-N1) respectively, the equal 10 μ g of every kind of plasmid, It is overexpressed 40h.

The construction method of pEGFP-P60 plasmid are as follows: extract Vero cell total rna, with 5'-ATGGCGGCAGGAGGC-3' with 5'-CAACTGGATCTCACG-3' is upstream and downstream primer, and reverse transcription obtains P60cDNA, carries out PCR amplification, PCR amplification is produced Object adds restriction enzyme site EcoRI and BamHI respectively；The carrier pEGFP-N1 that can stablize expression GFP is selected simultaneously, in the upstream EGFP Select restriction enzyme site EcoRI and BamHI.It is connected after p60 gene is distinguished double digestion with pEGFP-N1 carrier, building obtains PEGFP-P60 plasmid.Plasmid pEGFP-N1 map is shown in Fig. 6.

Virus inoculation: Transfected cells distinguish virus infection, the equal 10TCID of every kind of virus₅₀, cultivate different time.

7, Western blot, lesion method and RT-PCR method research P60 are overexpressed or knock out the influence to virus virulence

Immunoblotting: the pH7.5PBS for step 5, in 6 knocking out or being overexpressed the cell pre-cooling for handling and infecting is washed 3 times Afterwards, cell scraper scrapes, and behind total protein of cell 3-5 seconds that ultrasound cracking is extracted, mixes, boils with 6x SDS-PAGE sample-loading buffer 10min is boiled, SDS-PAGE electrophoresis is carried out, is transferred to the albumen on gel on pvdf membrane by electric transferring film instrument after electrophoresis.Containing 5% The PBST buffer room temperature of skimmed milk power closes lh, and 4 DEG C of shakes of primary antibody is added to be incubated for 2h.Film 4x5min is washed with PBST, l:8000 is dilute The corresponding secondary antibody room temperature for releasing HRP label, which is shaken, is incubated for 1h, washes film 4x5min, finally uses SuperSignal West Pico Chemilumineseent HRPSubstrate ECL colour developing, and with Kodak's medical film exposure, last developing fixing is seen It examines.Wherein antibody: virus protein antibody is purchased from Santa Cruz biotech company (Santa Cruz Biotechnology). It is that century biotechnology has that the fluorescence of Red-x containing Rhodamine goat anti-mouse lgG secondary antibody, which is purchased from Beijing health with other secondary antibodies, Limit company.Virus protein antibody used, including ICP8, ICP0, NP antibody and p60 antibody and actin antibody, are 1: 1000 dilutions.See Fig. 2A 2 and B2.It is replicated it can be seen that p60 knockout is unfavorable for HSV-1 and NDV, and both diseases can then be optimized by being overexpressed The duplication of poison.

Lesion tests (TCID₅₀/EID₅₀): the cell sample difference for handling and infecting is knocked out or is overexpressed by step 5, in 6 It collects, wherein MDV is digested with 0.25% pancreatin, other virus infected cells are using multigelation method harvest virus, i.e., will be thin Born of the same parents bottle be put into -20 DEG C freeze 2 hours after place room temperature to partial melting, yawing cell bottle makes attached cell take off wall at this time, again It is put into -20 DEG C to freeze, 3 times repeatedly, releases virus from cell.It, need to be -70 if all samples do not have to temporarily DEG C or liquid nitrogen in freeze.Then carry out, 1) cytopathy analysis: by the Hep-2 cell of HSV and NDV infection, AIV infects vero Cell, MDV infect the sample of CEF cell, carry out doubling dilution and obtain cell toxicant suspension, with 96 orifice plate culture CEF cells to list 50 hole μ L/ (including HSV, NDV, MDV and AIV) of cell toxicant suspension is added into each hole, does 12 dilutions and 8 repetitions for layer, Cytopathy is observed after placing 48 hours in 37 DEG C of carbon dioxide incubators, and calculates TCID₅₀(form the virus of cytopathy Titre), it is repeated 3 times.In addition, immunofluorescence analysis: the CFE cell culture medium of CDV infection, which is inhaled, to be abandoned, and after washing 3 times with PBS, adds 4% Formaldehyde it is fixed after, be incubated for 60min with CDV mouse monoclonal antibody, washed 3 times with PBS, Rhodamine Red-x label is added Mountain sheep anti mouse secondary antibody be incubated for 40min, washed 3 times with PBS.Under immunofluorescence microscopy 568nm wavelength, by observing and recording Virus fluorescence, to carry out toxicity test and calculate TCID₅₀。2)EID₅₀Analysis: the cell sample of IBV will be infected, 10 multiple proportions are dilute It releases, is inoculated with SPF chicken embryo, every 1 μ L of egg inoculation measures IBV chicken embryo median infective dose EID₅₀, according to Reed-Muench formula It calculates and determines above-mentioned 6 kinds of viral virulence.See Fig. 2A 1 and B1；Fig. 3 A2, B2, C2, D；Fig. 4 A2, B2, C2, D.

Real-time quantitative RT-PCR: by step 5, the cell sample infected in 6 is collected respectively, is said according to Trizol lysate Bright book extracts total serum IgE.Specific downstream primer is used to carry out the conjunction of cDNA as reverse transcription primer (using primer shown in step 4) At.Real-time quantitative PCR amplification is carried out with SYBR Green I dye method.20 μ L overall reaction systems: 10 μ 2 × SYBR of L Green I PCR mix, each 0.4 μ L of upstream and downstream primer (10 μM), moisturizing to 20 μ L.Real-Time PCR System (ViiA 7) setting Response procedures: 95 DEG C of initial denaturation 5min are denaturalized 95 DEG C of 10s, 60 DEG C of annealing and extension 45s, set 40 circulations, finally melted Solution curve analysis.Take 2^-ΔΔtMethod carries out relative quantification to target gene, and β-actin is selected as reference gene.See Fig. 3 A1, B1, C1；Fig. 4 A1, B1, C1.

8, immunoblotting, cell/chicken embryo lesion and RT-PCR method eligible result

Wherein immunoblotting partial results (HSV and NDV) are shown in Fig. 2.

Influence (RT-PCR method) about knockout p60 albumen to virus replication: CDV (tests average value) three times: RT-PCR The sip60 processing group measured is respectively as follows: 1.23,2.76,47.25 in 12,24,48 hours.The siNeg processing group that RT-PCR is measured, 12, it is respectively as follows: 2.97,11.13,173.0 within 24,48 hours.MDV (tests average value) three times: the sip60 processing that RT-PCR is measured Group is respectively as follows: 5.52,16.71,180.79 in 12,24,48 hours.The siNeg processing group that RT-PCR is measured, 12,24,48 hours It is respectively as follows: 11.39,45.47,429.56.IBV (tests average value) three times: the sip60 processing group that RT-PCR is measured, 12,24, It is respectively as follows: 15.15,32.5,91.2 within 48 hours.The siNeg processing group that RT-PCR is measured is respectively as follows: 29.9 in 12,24,48 hours, 86.5 205.6.

About influence (RT-PCR method) of the overexpression p60 albumen to virus replication: after visible infection 6 hours, difference is aobvious It writes, p < 0.01 * *.In Fig. 4 A1, B1, C1, with 2^-△△CTMethod calculates the difference of virus mRNA expression between different disposal group.With GraphPad prism5 draws.The N1 processing of each time point is all standardized as 1.Because gap is obvious, specific number is not arranged Out.

Influence (TCID about 60 albumen of overexpression to virus replication₅₀Method):

CDV----N1 processing group is TCID50=10^5.58/ ml, P60 TCID50=10^6.70/ml。

HSV----N1 is TCID50=10^8.3/ ml, P60 TCID50=10^10.52/ml。

NDV----N1 is TCID50=10^8.6/ ml, P60 TCID50=10^10.58/ml。

MDV---N1 is TCID50=10^4.29/ ml, P60 TCID50=10^5.60/ml。

IBV---N1 is EID₅₀=10^6.5/ ml, P60 EID₅₀=10^7.5/ml。

AIV---N1 is TCID50=10^5.2/ ml, P60 TCID50=10^7.1/ml。

To sum up, N1 and P60 is overexpressed to TCID after virus infection₅₀Influence difference between Ig 1-3, gap is obvious.

Influence (TCID about 60 albumen of knockout to virus replication₅₀Method):

CDV----N1 processing group is TCID50=10^6.38/ ml, P60 TCID50=10^4.58/ml。

HSV----N1 is TCID50=10^8.63/ ml, P60 TCID50=10^6.32/ml。

NDV----N1 is TCID50=10^8.92/ ml, P60 TCID50=10^6.78/ml。

MDV---N1 is TCID50=10^5.25/ ml, P60 TCID50=10^4.25/ml。

IBV---N1 is EID₅₀=10^7.5/ ml, P60 EID₅₀=10^6.25/ml。

AIV---N1 is TCID50=10^5.32/ ml, P60 TCID50=10^4.1/ml。

To sum up, N1 and P60 is knocked out to TCID after virus infection₅₀Influence difference between Ig 1-3, gap is obvious.

Result of study further proves that p60 albumen can optimize paramyxovirus, coronavirus, orthomyxovirus and herpesviral Duplication.

Fig. 1 is characteristic and function of the p60 in virus infection host cell, and Fig. 5 is the effect that p60 can optimize virus replication Mechanism.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (5)

1. glioma suppression cancer candidate gene 2 encode albumen preparing the application in inactivated vaccine and/or attenuated live vaccine, be by After the gene is overexpressed in host cell, then transfected virus culture；

Wherein, the glioma suppression cancer candidate gene 2 encodes the amino acid sequence of albumen as shown in Seq ID No.1；

The host cell is CEF, Hep-2, Vero；The inactivated vaccine and/or attenuated live vaccine are coronaviridae, pair is viscous The inactivated vaccine and/or attenuated live vaccine of Viraceae, orthomyxoviridae family and herpetoviridae.

2. the inactivated vaccine and/or attenuated live vaccine preparation method of a kind of optimization, which is characterized in that be overexpressed in host cell Glioma presses down cancer candidate gene 2, and is inoculated with host cell with inactivated vaccine and/or attenuated live vaccine production strain, and culture is thin Born of the same parents harvest virus liquid；

Wherein, the definition of the glioma suppression cancer candidate gene 2, host cell and inactivated vaccine and/or attenuated live vaccine is same Described in claim 1.

3. according to the method described in claim 2, it is characterized in that, when the convergence degree of host cell reaches 80~90%, by 1 ~10 TCID₅₀Strain is used in inoculation inactivated vaccine and/or attenuated live vaccine production.

4. according to the method described in claim 2, it is characterized in that, cell culture mode is adhere-wall culture or the culture that suspends.

5. the host cell for being overexpressed glioma suppression cancer candidate gene 2 is preparing answering in inactivated vaccine and/or attenuated live vaccine With；

Wherein, the definition of the glioma suppression cancer candidate gene 2, host cell and inactivated vaccine and/or attenuated live vaccine is same Described in claim 1.