CN109187982B

CN109187982B - Method for screening and identifying TLR vaccine adjuvant

Info

Publication number: CN109187982B
Application number: CN201810871161.6A
Authority: CN
Inventors: 杨文君; 其他发明人请求不公开姓名
Original assignee: Zhejiang Compvss Biotechnology Co ltd
Current assignee: Zhejiang Compvss Biotechnology Co ltd
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2021-06-04
Anticipated expiration: 2038-08-02
Also published as: CN109187982A

Abstract

The invention relates to the field of vaccines, and discloses a method for screening and identifying a TLR vaccine adjuvant, which comprises the following steps: A) splitting and pre-separating virus; B) SDS-PAGE separation and LC-MS identify specific virus subcomponent proteins; C) separating virus subfraction protein by an electroelution method; D) construction of 293T cell lines stably expressing B5R, D8L and K2L and determination of TLR2 ligand activity. The invention establishes a whole set of methods for screening and identifying the vaccinia virus protein subfractions with the activity of the TLR2 ligand, identifies that the virus protein D8L has the activity of the TLR2 ligand, and can be used for preparing TLR vaccine adjuvants for resisting virus infection and tumors.

Description

Method for screening and identifying TLR vaccine adjuvant

Technical Field

The invention relates to the field of vaccines, in particular to a method for screening and identifying a TLR vaccine adjuvant.

Background

The immune response mechanism of the host in response to viral infection recognizes related ligands in different viruses, mainly through Pattern Recognition Receptors (PRRs), which are called pathogen-associated molecular patterns (PAMPs), including viral proteins, DNA, RNA, etc. Unlike viral DNA and RNA, viral proteins are primarily recognized by TLR family proteins, including TLR2 and TLR 4. Innate immune cells, such as macrophages and DCs, both express TLR2 and TLR4 and are therefore activated by the associated virus and secrete a variety of inflammatory factors that lead to the activation of the host immune response. Our findings indicate that Vaccinia Virus (VV) can activate the TLR2-MyD88 signaling pathway in DC Cells (DCs) or macrophages, leading to increased secretion of pro-inflammatory factors and enhanced T cell responses. However, it is still unclear which molecule TLR2 is specifically activated by in the viral particle.

Vaccines play an important role in the prevention and control of infectious diseases. For example, vaccinia virus vaccines were used for the prevention of variola virus, making variola virus almost extinct since the last 70 s. Traditional vaccinia virus vaccines are mainly prepared from live vaccinia virus, often associated with high frequency of toxic and side effects, and even potentially harmful to patients with eczema and immunodeficiency. In contrast, a safer alternative is to use highly purified recombinant protein vaccines. With the development of novel recombinant protein vaccines, protein vaccines are also widely used in the treatment of other malignant diseases, such as in the treatment of tumors. The principle is that tumor antigen is introduced into a patient body in the form of tumor-related protein or polypeptide, so that the immunosuppression state caused by tumor is overcome, the immunogenicity is enhanced, the immune system of the patient is activated, and the cellular immunity and humoral immunity response of the organism are induced, thereby achieving the purpose of controlling or eliminating the tumor. Most vaccines studied today are based on highly purified recombinant proteins or pathogenic subunits. However, many recombinant proteins have a weak ability to activate a protective immune response, and therefore protein vaccines need to be used in combination with adjuvants to enhance the immunogenicity of antigens. An adjuvant is a non-specific immunopotentiator. It is generally thought that the kind of adjuvant directly determines the effect of immunization and the type of immune response, but the mechanism of action thereof is still unclear. It is imperative to improve the components of vaccines and adjuvants to enhance immune efficacy. In the case of vaccinia virus, which contains about 30 protein components in total, relevant studies have identified that many viral surface antigen genes function in host immune responses, but it is unclear by which mechanism these antigen proteins activate the host immune response and whether vaccine adjuvants are needed to achieve immune protection. Therefore, a complete set of technical methods for analyzing and identifying the protein subfractions with vaccine adjuvant activity in vaccinia virus based on certain specific indexes is needed, and a safer and more efficient vaccine adjuvant suitable for anti-virus and anti-tumor is developed based on the technical methods. .

Disclosure of Invention

In order to solve the technical problems, the invention establishes a method for screening and identifying the TLR vaccine adjuvant, and particularly establishes a whole set of technical methods for screening and identifying the vaccinia virus protein subfractions with the activity of the TLR2 ligand, and identifies that the virus proteins D8L and K2L have the activity of the TLR2 ligand. The TLR2 ligand of D8L has stronger activity, and the recombinant protein D8L alone is used for immunizing mice to enhance the immune response in the mice and generate protective antibodies. Therefore, the identified virus recombinant protein D8L is not only a TLR vaccine adjuvant, but also an immunogenic virus antigen, and can be used for preparing a protein vaccine for resisting smallpox virus infection. Since TLR2 causes the innate immunity or humoral immunity of the organism by recognizing various pathogen-related molecular patterns, the technology is also suitable for screening and identifying TLR vaccine adjuvants against other viruses or pathogens. In addition, tumor associated antigens carried by tumor cells can be recognized by TLR receptors, so that the TLR vaccine adjuvant can also be used for stimulating the immunocompetence of a human body to the tumor cells, and has an anti-tumor effect.

The specific technical scheme of the invention is as follows: a method for screening and identifying a TLR vaccine adjuvant comprises the following steps:

A) splitting and pre-separating virus;

B) SDS-PAGE separation and LC-MS identify specific virus subcomponent proteins;

C) virus subcomponent protein is separated by an electroelution method and is used for activity determination of a TLR2 ligand;

D) construction of 293T cell line stably expressing virus recombinant protein and determination of TLR2 ligand activity.

Preferably, the viral recombinant protein is B5R, D8L or K2L.

Pathogen-associated molecular patterns (PAMPs) on the surface of viruses can be recognized by receptors of the TLR family on the surface of host immune cells, resulting in an immune response in the host.

Previous studies by the present team have found that the enhancement of host immune responses by vaccinia virus, including DC activation and maturation, and T cell responses, all depend on the activation of the TLR2 pathway. Thus, by virtue of the activity assay of the TLR2 pathway, it is possible to analyze and identify which viral protein components are capable of activating the host immune response.

Therefore, on the basis, the invention establishes a whole set of technical methods for screening and identifying the vaccinia virus protein subfractions with the activity of the TLR2 ligand, and identifies that the virus proteins D8L and K2L have the activity of the TLR2 ligand, wherein the activity of the TLR2 ligand of D8L is stronger, and the immune response in mice can be enhanced and protective antibodies can be generated by immunizing the mice with the recombinant protein D8L alone. Therefore, the identified virus recombinant protein D8L can be used as an adjuvant of a protein vaccinia vaccine and a TLR vaccine at the same time. The technology is also suitable for screening and identifying other anti-virus and anti-tumor TLR vaccine adjuvants.

Preferably, step a) is specifically:

1) the vaccinia virus was lysed with the mild detergent Triton X-100 to obtain a viral lysate.

Triton X-100 is a mild non-denaturing extractant that effectively preserves biological activity.

2) Dialyzing with dialysis card with molecular sieve to remove detergent component in virus lysate.

3) The virus lysate was centrifuged to remove insoluble pellet and the protein concentration in the supernatant was determined.

4) The proteins are separated into three viral subfraction proteins of <30kD, 30-100kD and >100kD using molecular sieves.

5) And (3) respectively measuring the protein concentration of each virus subcomponent protein, and taking part of each virus subcomponent protein for activity measurement of the TLR2 ligand.

6) Determining TLR2 ligand activity for each viral subcomponent protein according to the method in step D), screening for viral subcomponent proteins having TLR2 ligand activity for subsequent isolation and identification steps.

Preferably, in step 3) of step A), the centrifugation conditions are 13000-17000g for 20-40 min.

Preferably, step B) is specifically:

1) separating 100-200 μ g of the viral subfraction protein isolated in step A) by non-reducing and non-denaturing SDS-PAGE.

The method can separate proteins according to molecular weight without destroying disulfide bonds in the protein structure, thereby protecting the possible antigenic properties of the proteins.

2) Staining by a Coomassie brilliant blue method, and distinguishing protein bands with different molecular weights;

3) photographing and cutting off each band, taking a part of gel block, digesting protein in the gel into polypeptide fragments by an in-gel enzymolysis method, and then identifying the polypeptide sequence and the protein type of each band by LC-MS;

4) the residual gel block is used for separating virus subcomponent protein by an electroelution method.

Preferably, step C) is specifically:

1) carrying out electric elution on the residual gel block in the step B) by using an electric elution instrument, wherein the electric elution conditions are as follows: keeping constant pressure of 55-65V at 1-5 deg.C overnight;

2) selecting a molecular sieve with the molecular weight of 30kD for dialysis, and removing virus subcomponent proteins below 30kD and buffer salt components in an electrophoresis buffer solution;

3) the virus subfraction protein is concentrated to 80-120 mu L by using a 10kD molecular sieve, and after the concentration is measured, the virus subfraction protein can be used for measuring the activity of the TLR2 ligand.

The protein separated by the method of the step B) retains most of the antigenic property of the protein, so the protein can be eluted from the gel by an electroelution method, and the cell expressing the TLR2 reporter gene is treated in vitro, thereby screening the viral protein subcomponent with the activity of the TLR2 ligand.

Preferably, step D) is specifically:

1) culturing and expanding HEK293 cells stably expressing human TLR2 or TLR4 in vitro: HEK293, HEK293-hTLR2/CD14, HEK293-hTLR4/CD14 and HEK293-hCD 14; HEK293 and HEK293-hCD14 served as negative controls that did not express TLR2 or TLR 4.

2) Diluting the virus subcomponent proteins separated in the step A) and the step C) to different concentrations, and stimulating the above cells respectively.

3) After 15-20h of stimulation, the secretion of human IL-8 in the supernatant is detected by using an ELISA kit, and after a TLR ligand in a culture medium is combined with TLR2 or TLR4 in HEK293 cells, an NF-kappa B signal channel is activated, and the expression and secretion of human IL-8 are increased, so that the activation condition of TLR2 can be reflected by detecting the content of human IL-8 in the supernatant.

4) Constructing an expression plasmid containing the virus subcomponent protein ORF from the virus subcomponent protein identified in the step B), and transfecting 293T cells to obtain 293T transgenic cells stably expressing the virus recombinant protein.

5) Co-culturing the obtained 293T transgenic cell with HEK293, HEK293-hTLR2/CD14, HEK293-hTLR4/CD14 and HEK293-hCD14 according to different concentrations respectively; collecting supernatant after culturing for 15-20h, and determining the content of human IL-8 in the supernatant according to the method in the step 3) for verifying whether certain virus protein expressed in cells has the activity of the TLR2 ligand.

Compared with the prior art, the invention has the beneficial effects that:

1. in the prior art, no screening and identification method of the vaccinia vaccine adjuvant based on the determination of the activity of the TLR2 ligand is available. According to the invention, researches show that the recombinant virus protein D8L with the activity of the TLR2 ligand can cause the enhancement of immune response in a mouse body and protective humoral immunity by singly immunizing the mouse, so that the antigen is a TLR adjuvant and has the immune protection effect on virus infection. According to the technical scheme, the specific virus protein component with the activity of the TLR2 ligand is screened and identified, and the protein vaccine and the TLR vaccine adjuvant capable of resisting smallpox virus infection can be prepared on the basis of the recombinant virus protein. In addition, since TLR2 can broadly recognize a variety of pathogen-associated molecular patterns and tumor-associated antigens, this method can be used to screen and identify TLR-like adjuvants against other types of viruses or tumors.

2. The method for separating and identifying the virus protein components comprises a virus splitting and pre-separating method, a method for separating and identifying virus proteins by combining SDS-PAGE and LC-MS, and a method for detecting the activity of TLR2 ligand of the virus proteins. The above isolation identification and detection methods, used in combination, can identify which specific viral protein subcomponent(s) have TLR2 ligand activity.

Drawings

Figure 1 is a graph of the results of a pre-isolation experiment of the VV protein fraction having TLR2 ligand activity of example 1 of the invention;

FIG. 2 is a diagram showing the results of an experiment for separating and identifying VV protein by SDS-PAGE in example 1 of the present invention;

FIG. 3 is a graph of the experimental results of the identification of D8L/K2L having TLR2 ligand activity of example 1 of the invention;

FIG. 4 is a graph showing the results of experiments in which recombinant D8L stimulated production of cytokines and promoted maturation of DCs, resulting in enhancement of T cell immune responses in example 1 of the present invention;

FIG. 5 is a graph showing the results of the experiment in which D8L induced mice to produce specific protective antibodies in example 1 of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples.

Introduction of proteins and cells:

B5R is the existing protein, and the amino acid sequence information is as follows:

>tr|B1PHX2|B1PHX2_9POXV B5R (Fragment) OS=Vaccinia virus OX=10245 PE=4 SV=1

MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDQGYHSSDPNAVCETDKWKYENPCKKMCTVSDYISELYNKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGASYISCTANSWNVIPSCQQKCDMPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPVLPICVRTNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIVALTIMGVIFLISVIVLVCSCDKNNDQYKFHKLLP

for details, reference may be made to the "Characterization of a vaccine Virus-Encoded 42-Kilodalton Class I Membrane microorganism Component of the excellular Virus Envelope".

D8L is the existing protein, and the amino acid sequence information is as follows:

>tr|A0A2I2MC48|A0A2I2MC48_VACCW IMV membrane protein OS=Vaccinia virus (strain Western Reserve) OX=10254 GN=D8L PE=4 SV=1

MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNEYVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGLIIISIFLQVLDHKNVYFQKIVNQLDSIRSANTSAPFDSVFYLDNLLPSKLDYFTYLGTTINHSADAVWIIFPTPINIHSDQLSKFRTLLSSSNHDGKPHYITENYRNPYKLNDDTQVYYSGEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIAIVFVFILTAILFFMSRRYSREKQN

for details, reference may be made to the "Structural and Biochemical Characterization of the vaccine viral Protein D8 and Its Characterization by the Antibody LA 5" and the "N-Terminal Amino Acid Sequences of vaccine viral Structural Proteins".

K2L is a conventional protein, and the amino acid sequence information is as follows:

>sp|P18384|SPI3_VACCW Protein K2 OS=Vaccinia virus (strain Western Reserve) OX=10254 GN=SPI-3 PE=1 SV=1

MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHRFGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDITKTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMTHFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHMTRDPLYIYKMFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFILFMGKVESP

for details, reference may be made to the document "A vaccinia serine protease inhibitor while preserved virus-induced cell fusion".

The 293T cell line is a conventional cell, which is designated 293T (ATCC)^® CRL3216^TM) (ii) a HEK293, HEK293-hTLR2/CD14, HEK293-hTLR4/CD14 and HEK293-hCD14 are also present cells.

General examples

A method for screening and identifying a TLR vaccine adjuvant comprises the following steps:

A) splitting and pre-separating virus:

2) Dialyzing with dialysis card with molecular sieve to remove detergent component in virus lysate;

3) centrifuging the virus lysate (13000-17000 g, 20-40 min), removing insoluble precipitate, and determining the protein concentration in the supernatant;

4) separating the protein into three virus subfraction proteins of <30kD, 30-100kD and >100kD by using a molecular sieve;

5) respectively measuring the protein concentration of each virus subcomponent protein, and taking part of each virus subcomponent protein for the activity measurement of the TLR2 ligand;

B) SDS-PAGE separation and LC-MS identification of specific viral subfraction proteins:

C) And (3) separating virus subcomponent proteins by an electroelution method, and determining the activity of the TLR2 ligand:

D) Construction of 293T cell lines stably expressing B5R, D8L and K2L and determination of TLR2 ligand viability:

2) Diluting the virus subcomponent proteins separated in the step A) and the step C) into different concentrations, and stimulating the various cells respectively;

3) after 15-20h of stimulation, the secretion of human IL-8 in the supernatant is detected by using an ELISA kit, and after a TLR ligand in a culture medium is combined with TLR2 or TLR4 in HEK293 cells, an NF-kappa B signal channel is activated and the expression and secretion of human IL-8 are increased, so that the activation condition of TLR2 can be reflected by detecting the content of human IL-8 in the supernatant;

4) constructing expression plasmids containing ORF of the proteins according to the virus subcomponent proteins identified in the step B) for transfecting 293T cells to obtain 293T transgenic cells stably expressing the virus recombinant proteins;

Example 1

Experiment A: the virus splitting and pre-separating method includes the following steps:

a VV-WR virus strain (VR-1354) was purchased from ATCC. 1mg VV virus pellet was dissolved in 10ml PBS containing 1% Triton X-100, and incubated at 37 ℃ for 1 hour to extract total protein.

To remove the effects of detergents, total protein needs to go through the following dialysis steps: the lysate mixed with total viral proteins was added to Slide-A-Lyzer dialysis card (2 kDa MWCO) (Thermo Scientific, cat. No. 87719) and dialyzed overnight at 4 ℃. The specific dialysis method comprises the following steps: dialyzing at 4 deg.C in 1xPBS buffer solution, and changing the dialysate every 3 hr for 3 times.

The mixture was centrifuged at 15,000g for 30min to remove insoluble precipitate.

The mixture was assayed for protein concentration by BCA assay (Bio-Rad, cat # C503021-0500).

A250. mu.g mixture was ultrafiltered through 30kD MWCO and 100kD MWCO (Millipore, Cat. UFC903096, UFC 910096) to obtain a mixture of three protein components of different molecular weights: <30kD, 30-100kD and >100 kD.

The protein concentration of the three protein subfractions was determined here by the BCA method.

Experiment B: a method for separating virus subfractions pre-separated in experiment A by SDS-PAGE comprises the following steps:

SDS-PAGE electrophoresis buffer preparation:

1 Xelectrode buffer (0.025mol/L Tris, 0.2mol/L glycine, 1% SDS, pH8.3): 3.02g Tris plus 18.8g glycine, SDS 10g, diluted to 1L with double distilled water. Can be stored at room temperature for one month.

2xSDS loading buffer (0.1 mol/L Tris-HCl, 2% SDS, pH 6.8): 1mL of 1mol/L Tris-HCl pH6.8, 0.4g SDS, 2mL glycerol, 1mL of 0.1% bromophenol blue, diluted to 10mL with double distilled water, can be stored at-20 ℃ for 6 months. Note that the loading buffer was not supplemented with reducing agents to avoid disruption of disulfide bonds in the protein.

SDS-PAGE electrophoresis:

and (3) cleaning the glass plate, the rubber pad and the comb by using double distilled water, wiping by using an alcohol cotton ball, and installing the electrophoresis tank.

The protein sample is added into the equal volume of 2xSDS loading buffer and mixed evenly. Note that the sample is not denatured by boiling, avoiding disruption of the higher order structure of the protein.

A new piece of 4-12% pre-made gel (Bio-rad, cat # 3450123) was removed, the package unpacked, the top comb carefully removed, the top layer of the gel was rinsed with double distilled water and the remaining water droplets were blotted with absorbent paper.

And (5) clamping the prefabricated gel into an electrophoresis tank. Add electrode buffer, slowly spot 500. mu.l sample (total amount between 100-200. mu.g) to the bottom of the well with a micro-sampler, and simultaneously spot 10. mu.l standard protein (Bio-Rad) as a molecular weight reference on the side wells, and perform electrophoresis at a constant voltage of 200V.

When the bromophenol blue reaches the separation gel, the voltage is changed to 250 volts, and electrophoresis is continued until the bromophenol blue reaches the bottom of the gel.

After the electrophoresis was completed, the gel was peeled off and placed in an appropriate amount of Coomassie brilliant blue staining solution (Thermo Scientific, cat. No. 24590) to ensure that the gel was sufficiently covered with the staining solution. The cells were placed on a horizontal shaker and slowly shaken and stained at room temperature for 1 hour.

Pouring out the staining solution, adding a proper amount of Coomassie brilliant blue destaining solution, slowly shaking on a horizontal shaking table, and destaining for more than 4 hours at room temperature until protein bands on the gel are clearly visible.

After the decolorization was completed, the plate was washed with pure water and photographed. A total of 9 bands between 30-100 kDa were excised, with reference to molecular weight standards.

The excised protein bands were cut into small pieces, and the excised gel pieces of each band were divided into two parts. One portion was used for the electroelution of experiment C to isolate viral proteins. The other part of the gel block was used for in-gel enzymatic digestion and LC-MS identification in experiment D.

Experiment C: the method for separating the virus protein by using the electro-elution method for detecting the activity of the TLR2 ligand comprises the following specific operation method:

the above 9 protein bands were electroeluted using an electroelution apparatus (Bio-rad) under the following conditions: 60V constant pressure 4 ℃ overnight.

The eluted sample was then transferred to Slide-A-Lyzer dialysis card (Thermo Scientific, cat # 66030) containing 30kDa molecular sieves, dialyzed overnight at 4 ℃ against 1,000 ml of 1XPBS buffer, and the solution was changed 1 time every 3 hours and 3 times during dialysis.

The sample was aspirated from the dialysis card and concentrated to a volume of 100 μ l using a 10kD MWCO ultrafiltration tube (Millipore, cat # UFC 901024).

The protein concentration of the concentrate was measured by BCA method (Bio-Rad, cat # C503021-0500).

These fractions were tested for TLR2 ligand activity using HEK293-hTLR2/CD14 cells. See experiment E for a specific procedure.

Experiment D: the in-gel enzymolysis and LC-MS identification of the protein subfractions of the virus are carried out by the following specific operation methods:

the chopped slab was placed in an EP tube (slab cut to about 1 mm)³Size).

Add 400. mu.l of 100mM NH₄HCO₃Decolorizing with 30% ACN, washing to transparent, removing supernatant, and lyophilizing.

Add 90. mu.l of 100mM NH to each tube₄HCO₃Mu.l of 100mM DTT was incubated at 56 ℃ for 30 minutes to reduce the protein.

The supernatant was removed and 100. mu.l of 100% ACN was added to each tube and aspirated after 5 minutes.

Add 70. mu.l of 100mM NH to each tube₄HCO₃30 μ l of 200mM IAA (ready-to-use, protected from light) and left in the dark for 20 minutes.

Remove supernatant and add 100. mu.l of 100mM NH per tube₄HCO₃Room temperature for 15 minutes.

The supernatant was removed, 100. mu.l of 100% ACN was added, and after 5 minutes, the mixture was aspirated and lyophilized.

After freeze-drying, 5 mul of 10 ng/mul of Trypsin solution is respectively added, and the mixture is placed in a refrigerator at 4 ℃ for 30-60 minutes to ensure that the gel block is fully imbibed.

30. mu.l of 50mM NH were added₄HCO₃Buffer solution (without Trypsin), PH 7.8-8.0.

The reaction was carried out at 37 ℃ overnight for about 20 hours.

Sucking out the protein enzymolysis solution, transferring to new EP tube, adding 100ul 60% ACN/0.1% TFA into original tube, performing ultrasonic treatment for 15 min, sucking out the solution, mixing with the previous solution, repeatedly extracting for 3 times, mixing, and lyophilizing.

Sample preparation was completed, double distilled water was added for redissolving, spotting was performed, and the polypeptide sequence in each band was analyzed by LC-MS mass spectrometry, a specific protocol reference (Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigher L, Fotsis T, Man M. femto sequencing of protein from polyacrylic amides gels by no-electrophoresis mass spectrometry. Nature 1996;379: 466-.

Experiment E: the activity of the TLR2 ligand of the protein component is detected by using HEK293 cell strain which stably expresses hTLR2/CD 14. HEK293-hTLR2/CD14 is a plasmid for transfecting co-expression TLR2 and CD14 in HEK293 and is specially designed for detecting the activity of human TLR 2. In the presence of TLR2 ligand, CD14 significantly enhances the recognition of a pathogen by TLR2, and activation of TLR2 results in activation of the intracellular NF-kB pathway and secretion of IL-8. Therefore, whether the TLR2 pathway is activated by specific small molecules in the culture medium can be judged by detecting the supernatant IL-8. Similarly, activation of human TLR4 can be verified by detecting IL-8 secreted by HEK293-hTLR4/CD14 cells. While HEK293-hCD14 served as control cells for the above two cells, the ligands associated with TLR2 and TLR4 did not activate the NF-kB pathway of the cells. The specific operation steps are as follows:

the following cells were cultured for this experiment: HEK293 (ATCC, CRL-1573), HEK293-hTLR2/CD14 (Invivogen, cat No. 293-hTLR2CD 14), HEK293-hTLR4/CD14 (Invivogen, cat No. hTLR4md2CD 14) and HEK293-hCD14 (Invivogen, cat No. 293-hmd2CD 14), the medium composition being: DMEM, 10% heat-inactivated FBS, 1% double antibody. The culture method is reported in the literature (Zhang P, Cox CJ, Alvarez KM, Cunningham MW. Cutting edge: cardiac myosin activities in amino acids proteins lipids TLRs J Immunol 2009;183: 27-31.).

In step 1, the cells are stimulated by different virus protein components with different bands separated by electroelution in experiment C or components with different molecular weights pre-separated in experiment A with different concentrations respectively. For the protein fraction of experiment C, the concentration in the medium was 250 ng/ml; for the total protein or protein fractions of different molecular weight of experiment A, 6 concentration gradients were set in the range of 250ng/ml and 7.8 ng/ml using a 2-fold dilution.

Supernatants were collected after 18 hours of stimulation with each of the above protein fractions, and the content of human IL-8 in the supernatants was measured by ELISA kit (BD bioscience, cat. 550999). The specific operation is according to the product specification.

Which viral protein components have TLR2 ligand activity can be determined by steps 2-3.

HEK293-TLR2/CD14 cells were co-cultured with 293T cells (293T-B5R, 293T-D8L and 293T-K2L) stably expressing B5R, D8L and K2L in experiment F in step 1. The ratio of the two co-cultured cells (293T: HEK293-TLR2/CD 14) was 1:32, 1:128, 1:512, 1:1024, respectively.

After 18 hours of co-culture, the supernatant was collected, and the content of human IL-8 in the supernatant was measured by ELISA kit (BD bioscience, cat. 550999). The specific operation is according to the product specification.

Which viral protein has TLR2 ligand activity can be judged by steps 2-3.

Experiment F: construction of 293T cell line stably expressing B5R, D8L and K2L. According to the results identified in experiment D, envelope proteins B5R, D8L and K2L were selected as candidates for viral protein components having TLR2 ligand activity, and 293T cells stably expressing these specific envelope proteins were constructed using gene cloning and transfection methods. These cells can be used for co-culture with HEK293-TLR2/CD14 cells in experiment E to determine the activity of the TLR2 ligand of the envelope protein. The specific operation method comprises the following steps:

the ORF sequences of the viral envelope proteins B5R, D8L and K2L were obtained from the NCIB database, and the terminator was removed. And designing primers according to sequences at two ends of ORF.

Carrying out PCR amplification under the following conditions: 1 ng of VV-WR DNA was used as a substrate template, and 7 viral gene primers were used for amplification. The reaction conditions were 95 ℃ for 1 cycle at 5 min. Denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 45-135s (1 kb/60s depending on gene size) for 30 cycles. Finally, extension was carried out at 72 ℃ for 7 min.

Sequencing the amplified product and determining the sequence is consistent with the record in the database.

The amplified product was digested with Not I (NEB, cat # R0189) and Kpn I (NEB, cat # R0142). Mammalian expression plasmids of recombinant B5R, D8L and K2L proteins with His-tag were constructed by ligating the cleaved products with T4 DNA ligase (NEB, cat # M0202) to NotI and Kpn I cleaved pBudCE4.1 vectors (Thermo Scientific, cat # V532-20), respectively.

293T cells were purchased from ATCC (CRL-3216). Cultures were grown in DMEM complete medium (DMEM +10% heat-inactivated FBS +1% double antibody). When the cells in the culture dish reached 80-90% density, 293T cells were transfected with the B5R, D8L and K2L expression plasmids constructed in step 5 according to the instructions of the transfection reagent (Thermo Scienfic, cat # 11668027).

The fresh medium was replaced 16 hours after transfection and 200. mu.g/ml Zeocin (Thermo Scienfic, cat # R25001) was added and 293T cells stably expressing VV protein were selected and named 293T-B5R, 293T-D8L and 293T-K2L.

Results of the experiment

As shown in figure 1, a component of VV protein with TLR2 ligand activity. HEK293-CD14 and-hTLR 2/CD14 cells were stimulated with different concentrations of the VV protein fraction ranging from 7.8 ng/ml to 250ng/ml for 18 hours, (A) total VV protein, (B) <30kD VV protein, (C) 30-100kD VV protein and (D) >100kD VV protein. The secretion of human IL-8 in the supernatant was measured by ELISA. Results are expressed as mean ± SD.

As shown in FIG. 2, VV protein was separated and identified by SDS-PAGE. (A) SDS-PAGE results of VV proteins. Left lane, molecular weight marker; right lane, total VV protein. (B) 9 protein bands, cut from the gel, varying in molecular weight from 30 to 100 kD. The proteins on the gel were subjected to electroelution and dialysis steps and used for in vitro stimulation. HEK293-TLR2/CD14 cells were stimulated with isolated viral proteins. After 18 hours, the supernatant was analyzed for secretion of human IL-8 by ELISA. Results are expressed as mean ± SD.

As shown in figure 3, D8L/K2L with TLR2 ligand activity. (A) A mammalian expression vector for expressing viral proteins, consisting of the full-length VV gene and V5-His tag. (B) Stably expressing (B5R, K2L, and D8L) cell lines were constructed in 293T cells. The cells were co-cultured with HEK293-TLR2/CD14 cells, respectively, and the ratio of the two cells is shown in the figure. (C) In addition, the supernatants of the above cells were added to the medium of HEK293-TLR2/CD14 cells for stimulation. After 18 hours, the supernatant was analyzed for secretion of human IL-8 by ELISA. Results are expressed as mean ± SD.

As shown in fig. 4, recombinant D8L stimulated the production of cytokines and promoted maturation of DCs, resulting in an enhanced T cell immune response. From (A) C57BL/6, (B) wild type C57BL/6 (WT), TLR2^-/-And MyD88^-/-DCs were isolated from bone marrow cells of mice and cultured for 5 days under GM-CSF and IL-4 stimulation. On day 5, DCs were stimulated with different concentrations of recombinant D8L protein for 18 hours, while recombinant a33R protein was added to the control group. Analysis of the culture supernatants by ELISASecretion of IL-6 and TNF- α. Results are expressed as mean ± SD. (C) DCs isolated from C57BL/6 mouse bone marrow cells were cultured for 5 days under stimulation with GM-CSF and IL-4. On day 5, DCs were stimulated with D8L for 18 hours, with a33R as a control. CD11c positive DCs were stained with CD11c and CD80/86 antibodies and analyzed by flow analysis for CD80/86 expression. The expression level of CD80/86 on the surface of CD11 c-positive DCs was reflected in Mean Fluorescence Intensity (MFI), as shown. (D) Total 2x10⁴Purified initial OT-1 CD8⁺T cells (CD 45.2)⁺) And 5X 10⁵OVA polypeptide-stimulated immature DCs were adoptively transferred together into congenic C57BL/6 mice (CD 45.1)⁺) In (1). The recipient mice were then injected subcutaneously with 10 μ g of recombinant D8L, along with a33R as a control. After 7 days, spleens of mice were removed to analyze the status of cells after transfer. Spleen cells were treated with 5. mu.g/ml brefeldin A and 2. mu.g/ml OVA polypeptide (I) (in vitro)²⁵⁷SIINFEKL²⁶⁴) After 6 hours of stimulation, intracellular staining was performed with anti-CD8 antibody, anti-CD45.2 antibody and anti-IFN-. gamma.antibody. Clonoid OT-1 CD8 in Total lymphocytes⁺The ratio of cells is shown in Panel D (top), CD8⁺The proportion of IFN-. gamma.positive clonal-like cells in T lymphocytes is shown in Panel D (bottom). Representative data from three independent experiments were selected for display.

As shown in fig. 5, D8L induced mice to produce specific protective antibodies. C57BL/6 mice were immunized with recombinant D8L or a33R (negative control) protein, sera were collected after 21 days of the last immunization, and titers of IgG1(a), IgG2 (B), IgG3(C) and neutralizing antibodies (IC 50) (D) specific for D8L were detected by flow cytometry. (A-C) the ordinate is OD450 and the abscissa is dilution, which shows that the serum content of IgG1, IgG2 and IgG3 in the D8L immunized mice decreases with increasing dilution, while the A33R immunized mice do not induce specific protective antibodies. (D) On the ordinate, IC50, i.e. serum dilution, it can be seen that the IC50 of the neutralizing antibodies induced by D8L is about 200, whereas a33R does not induce neutralizing antibodies.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for screening and identifying a vaccinia vaccine adjuvant, which is characterized by comprising the following steps:

A) splitting and pre-separating virus:

1) lysing the vaccinia virus with a mild detergent Triton X-100 to obtain a virus lysate;

3) centrifuging the virus lysate, removing insoluble precipitates, and measuring the protein concentration in the supernatant;

6) determining TLR2 ligand activity for each viral subcomponent protein according to the method in step D), screening for viral subcomponent proteins having TLR2 ligand activity for subsequent isolation and identification steps; B) SDS-PAGE separation and LC-MS identify specific virus subcomponent proteins;

2. The method of claim 1, wherein the recombinant viral protein is B5R, D8L or K2L.

3. The method for screening and identifying a vaccinia vaccine adjuvant according to claim 1, wherein in step 3) of step A), the centrifugation conditions are 13000-17000g for 20-40 min.

4. The method for screening and identifying a vaccinia vaccine adjuvant of claim 1, wherein step B) is specifically:

1) separating 100-200 μ g of the virus subfraction protein separated in step A) by non-reducing and non-denaturing SDS-PAGE;

5. The method for screening and identifying a vaccinia vaccine adjuvant of claim 4, wherein step C) is specifically:

6. The method for screening and identifying a vaccinia vaccine adjuvant of claim 5, wherein step D) is specifically:

1) culturing and expanding HEK293 cells stably expressing human TLR2 or TLR4 in vitro: HEK293, HEK293-hTLR2/CD14, HEK293-hTLR4/CD14 and HEK293-hCD 14; HEK293 and HEK293-hCD14 as negative controls that do not express TLR2 or TLR 4;

4) constructing an expression plasmid containing the virus subcomponent protein ORF according to the virus subcomponent protein identified in the step B), and transfecting 293T cells to obtain 293T transgenic cells stably expressing the virus recombinant protein;