CN113773371B

CN113773371B - VP1 protein, and preparation method and application thereof

Info

Publication number: CN113773371B
Application number: CN202111086545.5A
Authority: CN
Inventors: 吴洪流; 胡广; 张明; 郭蕾; 陈国�
Original assignee: Beijing Kyning Bioscience Co ltd
Current assignee: Beijing Kyning Bioscience Co ltd
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2024-06-18
Anticipated expiration: 2041-09-16
Also published as: CN113773371A

Abstract

The invention discloses VP1 protein, and a preparation method and application thereof. A preparation method of VP1 protein comprises the steps of washing, urea dissolving, denaturation and purification and renaturation of VP1 inclusion body protein induced to be expressed to obtain VP1 protein; wherein, the renaturation adopts an eddy renaturation device to carry out eddy renaturation. According to the invention, VP1 protein which is expressed by escherichia coli and does not have any redundant sequence is selected as an antigen, the expressed protein is renatured by using a vortex device, the protein is more similar to natural protein due to the conformational change rule of the protein under the action of uniform shearing force in the vortex renaturation device, the obtained antigen protein related detection indexes all accord with the human vaccine standard, the renatured protein and an aluminum adjuvant are mixed to immunize a mouse, a good immunogenicity reaction is generated in vivo, and the obtained serum has a higher neutralization titer, so that a foundation is laid for the development of EV71 or CA16 subunit vaccine.

Description

VP1 protein, and preparation method and application thereof

Technical Field

The invention relates to the biomedical field, and relates to VP1 protein, and a preparation method and application thereof.

Technical Field

Hand-foot-mouth disease is a common pediatric disease mainly characterized by fever, rash, ulcers and herpetic pharyngitis occurring in parts such as hands, feet, oral cavity and the like, and few patients can also cause myocarditis, pulmonary edema, encephalitis and other fatal complications, and the disease is frequently generated in infants under 5 years old. The pathogens causing hand-foot-and-mouth disease are mainly enterovirus type 71 (EV 71) and coxsackievirus type a16 (CA 16).

Studies have shown that EV71 and CA16 are mainly associated with severe hand-foot-and-mouth disease, which has neurotoxicity and further causes complications such as aseptic encephalitis, meningoepithymen encephalitis and myocarditis, and serious patients may be disabled or fatal.

EV71 and CA16 belong to picornaviruses, the genome of which is composed of 7408 nucleotide single-strand positive-strand RNA, encoding a 2194 amino acid polyprotein, which can be further hydrolyzed into three P1, P2 and P3 precursor proteins, the P1 precursor proteins encode VP1, VP2, VP3 and VP4 structural proteins, which together form the ionic shell of the virus, wherein VP1 directly determines the antigenicity of the virus and has genetic diversity completely corresponding to the virus serotypes, so that great importance is paid in the genome of EV71 and CA16 and vaccine development.

At present, no specific medicine exists for EV71 and CA16, so the development of vaccines is urgent. Because basic researches on etiology, infection mechanism, pathogenic mechanism, epidemiology and the like of EV71 and CA16 are not enough, EV71 and CA16 virus strains are easy to mutate in the epidemic process, and no good animal model exists, so that the difficulty of vaccine research and development is greatly increased. At present, the EV71 and CA16 vaccines mainly comprise inactivated vaccines, attenuated live vaccines, viroid particle vaccines, subunit vaccines, transgenic oral vaccines and synthetic peptide vaccines, although the inactivated viral vaccines are in clinical III test stage, the safety of the inactivated viral vaccines is yet to be verified, other vaccines are in research and development and clinical trial stages, and products on the market are not seen, so that the inactivated viral vaccines need to be put into the teams for vaccine development with great force.

The subunit vaccine of EV71 and CA16 mainly takes VP1 as a first-choice target, related researches show that antigens capable of generating better neutralizing antibodies are mainly VP1 proteins obtained by insect cell expression purification, and the general neutralizing antibodies with VP1 proteins expressed by escherichia coli as antigens have lower titers, and the main reasons are that the proteins expressed by escherichia coli are poorer in folding and processing, but the proteins of VP1 expressed by escherichia coli have the characteristics of high expression efficiency, low production cost and easy mass production, and the proteins obtained from escherichia coli have higher safety as vaccines and are easy to accept, so that subunit vaccine researches obtained from escherichia coli expression are becoming more and more favored.

Disclosure of Invention

The invention aims to overcome the defect of low titer of neutralizing protective antibodies generated by the existing EV71 and CA16 subunit vaccines, and provides a preparation method of EV71 or CA16 subunit vaccines which are renatured by using a vortex device, wherein VP1 protein of EV71 or CA16 is used as an antigen, renatured is carried out in the vortex device, and specific neutralizing antibodies are generated by the renatured proteins and aluminum adjuvant mixed immunized mice.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

A preparation method of VP1 protein comprises the steps of washing, urea dissolving, denaturation and purification and renaturation of VP1 inclusion body protein induced to be expressed to obtain VP1 protein; wherein, the renaturation adopts an eddy renaturation device to carry out eddy renaturation; the VP1 protein is selected from VP1 protein of EV71 or CA 16.

The VP1 sequence of EV71 is optimized through complete gene synthesis of a company, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the VP1 sequence of CA1671 provided by the invention is optimized through total gene synthesis of a company, the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; cloning the obtained VP1 nucleotide sequence into a pET22b expression vector to construct a pET22b-VP1 recombinant expression vector, introducing the plasmid into escherichia coli BL21trxB (DE 3) for induced expression, and obtaining VP1 inclusion body protein through ultrasonic centrifugation of the obtained bacterial liquid.

As one preferable mode of the preparation method, the inner diameter of a glass tube of the vortex renaturation device is 1cm, the length of the glass tube is 16cm, the liquid loading amount is 1mL, the angle of the renaturation glass tube is 30-80 degrees, the rotating speed is 3000-8000rpm, and the renaturation time is 10-30min.

As one preferable mode of the invention, the washing is to wash the VP1 inclusion body protein with a washing buffer solution 1, a washing buffer solution 2 and a washing buffer solution 3 in sequence, and finally, to rinse with ultrapure water, wherein the finally obtained protein is the washed inclusion body protein; wherein,

The formula of the washing buffer solution 1 is 20mmol/L Tris-HCl pH8.0,0.15mol/LNaCl,2mol/L urea, 1% TritonX-100,5mmol/L EDTA,

The formula of the washing buffer solution 2 is 20mmol/L Tris-HCl pH8.0,0.15mol/LNaCl,4mol/L urea, 1% TritonX-100,5mmol/L EDTA;

The formula of the washing buffer solution 3 is 20mmol/L Tris-HCl pH8.0,0.15mol/L LNaCl,2mol/L urea, 1% TritonX-100,5mmol/L EDTA and 2% sodium deoxycholate.

As one preferable preparation method of the invention, the urea dissolution is to dissolve the washed inclusion body protein with a high-concentration urea solution, and the supernatant obtained by centrifugation is the inclusion body protein after urea dissolution, wherein the high-concentration urea solution has a formula of 20mmol/L Tris-HCl pH of 8.0,8mol/L urea and 70mmol/L beta-mercaptoethanol.

As a preferred mode of the present invention, the preparation method, the denaturation and purification are carried out by subjecting the inclusion body protein to strong anion exchange purification and Sepharose CL-4B gel filtration chromatography in this order.

As one preferable mode of the preparation method, the strong anion exchange purification is carried out by using a strong anion exchange column filler which is pre-filled with acid-base rinsing to carry out strong anion exchange purification on the protein after inclusion body dissolution; preferably, the method comprises the steps of flushing a column with ultrapure water, then using an equilibration buffer until a base line is equilibrated, slowly loading a VP1 protein sample, flushing the column with the equilibration buffer again until the base line is equilibrated, eluting the equilibrated sample with an elution buffer, and purifying to obtain target protein; the formula of the balance buffer solution is 20mmol/L Tris-HCl pH8.0,8mol/L urea and 70mmol/L beta-mercaptoethanol; the elution buffer solution formula is 20mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol, 80mmol/LnaCl; the method for the gel filtration chromatography of the Sepharose CL-4B comprises the steps of carrying out PEG20000 dialysis bag embedding concentration on a VP1 protein sample subjected to strong anion exchange purification, carrying out the gel filtration chromatography purification analysis of the concentrated sample, wherein the concentration of the concentrated sample is 1.0-1.5 mg/ml.

As one preferable mode of the preparation method, the purified and denatured protein is subjected to rapid dialysis for 2-5 hours before renaturation, the dialyzate is 20mmol/L Tris-HCl,30-60mmol/L arginine, 5mmol/L EDTA,300mmol/L sodium chloride and 0.2mmol/L DTT, 1mL concentration of the dialyzed protein solution is 0.2-0.4mg/mL, and the renaturation is carried out by filling a glass tube with the length of 16cm and the diameter of 1cm, wherein the angle of the glass tube is 30-60 degrees, the rotation speed is 3000-6000rpm, and the renaturation time is 10-400min.

As a preferred aspect of the present invention, the preparation method comprises concentrating the vortex renatured protein solution and dialyzing with 5-200mmol/L HEPES buffer solution at pH 7.0.

EV71 and CA16 VP1 proteins prepared by the preparation method disclosed by the invention.

The EV71 and CA16 VP1 protein is applied to preparation of EV71 and CA16 subunit vaccines.

An EV71 and CA16 subunit vaccine comprising the EV71 and CA16 VP1 protein of claim 9 and an aluminum adjuvant.

The inclusion body protein is washed and centrifuged for multiple times by urea with different concentrations and surfactant, and finally the inclusion body is dissolved by urea with high concentration. The dissolved inclusion body protein can be purified by strong anion exchange under the existence of urea to obtain the protein with higher purity, and the purity of the antigen protein obtained after further purification by gel filtration chromatography is more than 95%. And (3) quickly dialyzing the purified urea-dissolved denatured protein in a dialyzate, putting the protein into a glass tube of a vortex device, and rotationally renaturating to obtain the subunit vaccine of EV71 and CA16 by mixing the protein and an aluminum adjuvant.

The beneficial effects are that:

According to the invention, VP1 protein which is expressed by escherichia coli and does not have any redundant sequence is selected as an antigen, the expressed protein is renatured by using a vortex device, the protein is more similar to natural protein due to the conformational change rule of the protein under the action of uniform shearing force in the vortex renaturation device, the obtained antigen protein related detection indexes all accord with the human vaccine standard, the renatured protein and an aluminum adjuvant are mixed to immunize a mouse, a good immunogenicity reaction is generated in vivo, and the obtained serum has a higher neutralization titer, so that a foundation is laid for the development of EV71 and CA16 subunit vaccines.

The inclusion body protein is washed by low-concentration and medium-concentration urea, wherein TritonX-100 and sodium deoxycholate are also added, most of host bacteria foreign proteins are removed in the washing process, and convenience is provided for subsequent purification.

The purified antigen protein is characterized in that the protein structure is completely opened in the presence of urea and mercaptoethanol, and a lot of proteins are adsorbed on a column and can be eluted by low-concentration salt ions during the purification by strong anion exchange, so that the protein polymerization degree is very low, even a lot of proteins are possibly in a monomer form, a vortex renaturation device is selected for renaturation of the protein, the conformation of the protein is more similar to that of natural proteins under the action of uniform shearing force, and an immune mouse is combined with an aluminum adjuvant, so that the immunogenicity and immunoreactivity of the antigen protein are greatly enhanced, and the obtained antibody titer and the neutralizing antibody titer are relatively high.

Compared with dialysis renaturation, the protein renaturation method has the advantages of short renaturation time requirement and high renaturation force, the obtained protein antigen part is more fully exposed, and a stronger protection effect is shown in a subsequent immune experiment. The vortex renaturation device used in the invention has strict requirements on the inner diameter and the length of the glass tube, and has certain requirements on the renaturation volume, the rotation speed, the angle of the renaturation glass tube and the renaturation time, and liquid is easy to splash when the volume is too large or the rotation speed is too high, so that reasonable use of the vortex renaturation device is the key of successful protein renaturation.

Compared with EV71 and CA16 inactivated vaccines in the current clinical test, the human enterovirus 71 and coxsackie 16 subunit vaccine provided by the invention has the characteristics of low cost, simplicity in operation, high safety, easiness in large-scale production and the like in the production process, and can generate better immunogenicity in vivo, and the obtained neutralizing antibody has higher titer, so that the vaccine is an alternative vaccine with potential clinical application value.

Drawings

FIG. 1 shows the cleavage verification of EV71 pET22b-VP1 recombinant vector

DNA standard molecular weight; before cleavage of pET22b-VP 1; single cleavage of pET22b-VP1 NdeI; pET22b-VP1 NdeI and SalI double cleavage.

FIG. 2 shows EV71 VP1 protein-induced expression

1. Protein standard molecular weight; 2. before induction; 3. after induction; 4. crushing the supernatant; 5. crushing the precipitate.

FIG. 3 shows ion exchange purification and gel filtration chromatography purification of EV71 VP1 protein

1. Protein standard molecular weight; 2. after the inclusion body urea is dissolved; 3. ion exchange purification; 4. purifying by gel filtration chromatography.

FIG. 4 shows the results of antibody titer detection

A is the detection result of the EV71VP1 protein antibody titer; b is the detection result of CA16 VP1 protein antibody titer

FIG. 5 shows detection of neutralizing antibody titer by EV71 micro-neutralization method

FIG. 6 is a photograph of an overall picture of an eddy current apparatus, the apparatus comprising two parts: and rotating the glass tube and the control box.

FIG. 7 is a photograph of a photographic element of an eddy current apparatus

A, rotating glass tube close-up, wherein the rotating glass tube consists of a motor, a coupling, a self-aligning bearing, a glass tube seat, a clamping device, a compression nut, an adjusting arm and a base.

And B, a local photo of the control box, wherein the outer surface of the control box consists of a rotating speed display, a speed regulating device, a power switch light and a 16-pin socket. The control box is internally composed of a 36-volt power supply and a motor driver, and is conventional equipment.

When in use, the power socket and the 16-pin socket connected between the glass tube rotating equipment and the control box are firstly inserted, the glass tube is inserted into the clamping device, the glass tube seat is put in, then the compression nut is screwed, and the angle between the glass tube and the plane is adjusted through the adjusting arm.

Next, the power switch on the control panel is pressed, at this time, the display speed on the rotation speed display is 0, and the rotation speed of the device is adjusted by clockwise rotation of the speed adjusting knob, and the number on the display is gradually increased along with the gradual increase of the number when the speed is not too fast in the speed adjusting process. The device can continuously run under the condition of 5000 revolutions, if higher rotation speed is needed, the heating condition of the bearing and the motor needs to be noted, the highest rotation speed can reach 11000 revolutions, and when the rotation speed is about 8500 revolutions, a resonance point is basically reached, the shaking of the device can be particularly severe, and the device can not run for a long time at 8500 revolutions as much as possible.

FIG. 8 construction of CA16 VP1 results in the cloning vector selection pET22b, and the cleavage sites selection NdeI and SalI.

FIG. 9 CA16 VP1 purification results

1: Inclusion body protein, 2: molecular sieve purification results, 3: q column purification

Detailed Description

The invention is further described below with reference to examples and figures.

Synthesis of the VP1 Gene and construction of the recombinant vector pET22b-VP1

EV71 and CA16 strains from C4 subtype are obtained from Shanghai Pasteur research, virus RNA is obtained by extracting with a virus RNA extraction kit (Tiangen Biotechnology Co., ltd.), virus cDNA is obtained by reverse transcription with a reverse transcription kit, the complete sequence of the virus is amplified by designing a primer segment, the segment sequence is cut and connected into complete sequence, the complete sequence is sent to Shanghai Yingjun Biotechnology Co., ltd for sequencing, the sequence of EV71 VP1 is finally detected as shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2, and the full-length cDNA sequence is also repackaged into a new virus with infectivity by in vitro transcription, so that the cDNA sequence is complete and the function is not lost.

The VP1 sequence obtained by the sequencing is used as a standard, the full gene sequence of VP1 is synthesized in Meite and biotechnology limited company, and since an initiation codon exists at the NdeI end of pET22b to start protein synthesis, an NdeI enzyme cutting site is added at the 5' end, a SalI enzyme cutting site is added at the 3' end and a termination codon is added at the 3' end during VP1 design, so that the expressed VP1 protein does not have any redundant sequence, pET22b is used as a skeleton plasmid, and pET22b and VP1 are respectively subjected to double enzyme cutting by NdeI and SalI, and the system is as follows:

The mixture was gently mixed, collected by centrifugation at the bottom of the tube, and after 3h in a 37℃water bath, transferred to a 80℃water bath for 20min. Taking 5 mu l of double enzyme-digested products to carry out agarose gel electrophoresis of 1.0%, using the rest enzyme-digested products to carry out agarose gel electrophoresis recovery, and measuring the concentration of the purified recovery products by a multifunctional enzyme-labeled instrument. The product fragments recovered after purification were ligated overnight with T4 ligase (TaKaRa) at 16℃with the following ligation system:

After overnight ligation at 16℃and transformation of the ligation product with 100. Mu.l E.coli DH5a, overnight incubation, colony PCR was performed to verify whether the clone was a positive clone, the positive clone was verified to extract the plasmid, and single or double digestion with Nde I and Sal I was performed to verify whether ligation was successful (FIG. 1), which indicated that recombinant vector pET22b-VP1 was constructed successfully.

2. Recombinant vector pET22b-VP1 transformed expression strain induced expression

E.coli BL21 trxB (DE 3) competent cells are transformed by the recombinant vector constructed successfully, meanwhile, colony PCR identification and enzyme digestion identification are carried out on colonies, and positive clones are induced to express. Single colonies were picked and incubated at 37 ℃ overnight at 1:50 (v/v) transferring into 100ml LB culture medium containing ampicillin (50. Mu.g/ml), culturing until OD ₆₀₀ is about 0.6, taking 1ml bacterial liquid into a 1.5ml centrifuge tube, centrifuging for 5min at 12000r/min, discarding supernatant, adding IPTG into the rest bacterial liquid to a final concentration of 0.5mmol/L, and inducing expression at 37 ℃ for 5 h. After induction of expression, the bacterial liquid was centrifuged at 10000rpm at 4℃for 10min, the bacterial cells were collected, and after sonication, 100. Mu.l of the disrupted liquid was centrifuged to obtain a disrupted supernatant and a disrupted pellet, and the bacterial cells before and after induction were added to the loading buffer, and SDS-PAGE analysis (FIG. 2) was performed, which revealed that all the expressed proteins were inclusion body proteins, and that the target protein expression amount was about 100mg/L.

Washing of VP1 inclusion body proteins

The method comprises the steps of carrying out ultrasonic disruption on 500ml of bacteria liquid for inducing expression, centrifuging to obtain inclusion body proteins, washing the obtained inclusion body with 2mol/L of low-concentration urea, wherein the washing buffer is 25ml (20 mmol/L Tris-HCl pH8.0,0.15mol/LNaCl,2mol/L urea, 1% Triton X-100,5mmol/L EDTA), ice bath conditions are washed twice, each time for 1.5h, centrifuging to remove supernatant, then adding 25ml (20 mmol/L Tris-HCl pH8.0,0.15mol/LNaCl,4mol/L urea, 1% Triton X-100,5mmol/L EDTA) into the precipitate, washing buffer is twice under ice bath conditions, each time for 1.5h, centrifuging to remove supernatant, adding 25ml (20 mmol/L Tris-HCl pH8.0,0.15 mol/Cl, 2mol/L urea, 1% Triton X-100,5mmol/L EDTA), centrifuging to remove sodium sulfate, washing the inclusion body is carried out after centrifugation for 1.5h, namely, dissolving the inclusion body proteins, namely, slightly dissolving the inclusion body proteins, namely, washing the inclusion body proteins are washed with ultra-pure water, namely, after centrifugation is carried out for 1.5h, the inclusion body is washed with 4 mmol/L of ultra-pure water, and finally, the inclusion body proteins are washed with 20 ml (20 mmol/5 ml) and then washed by centrifugation is washed.

Strong anion exchange purification of VP1 inclusion body proteins

And (3) pre-filling strong anion exchange column packing (Q SEPHAROSE FAST FLOW) subjected to acid-base rinsing (national institute of biochemical engineering technology), and carrying out strong anion exchange purification on the protein after inclusion body dissolution, wherein the protein has a volume of 25ml, a concentration of about 3mg/ml and an ion exchange purification packing dosage of about 50 ml. The column was rinsed with a large amount of ultrapure water and then equilibrated with equilibration buffer (20 mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol) until baseline equilibration, 25ml protein sample was slowly loaded and the column was rinsed again with equilibration buffer until baseline equilibration, where no protein flow-through was collected and almost all protein bound. The equilibrated sample was eluted with elution buffer (20 mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol, 80 mmol/LNaCl) to obtain the target protein (see FIG. 3), the protein purity was 95% or higher, the protein was equilibrated with this eluate, and then eluted with elution buffer (20 mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol, 130 mmol/LNaCl) having a slightly higher salt ion concentration, and after electrophoresis, the target protein content was higher, but the protein purity was significantly reduced, and almost all of the protein was eluted, and then washed with 1mol/L NaCl to almost eliminate the protein, so that finally the protein eluted at 80mmol/LNaCl concentration was defined as the harvest protein to obtain 75ml volume, and the protein concentration was about 0.5mg/ml.

Sepharose CL-4B gel filtration chromatography purification of VP1 inclusion body proteins

The ion-exchanged and purified sample is subjected to PEG20000 dialysis bag embedding concentration, the concentration of the concentrated sample is about 1.2mg/ml, the volume is about 25ml, and the gel filtration chromatography purification analysis is performed on the concentrated sample. The column was first rinsed with a large amount of ultrapure water, then equilibrated with equilibration buffer (20 mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol) until baseline equilibrated, the ion-exchanged purified sample was slowly loaded, the column was rinsed with equilibration buffer, the corresponding elution peak was collected, the final elution volume was about 50ml, the protein concentration was about 0.5mg/ml, and SDS-PAGE analysis was performed, as shown in FIG. 3.

Vortex renaturation after washing and purifying VP1 inclusion body protein

The protein concentration of the denatured and purified EV71 VP1 is 0.7mg/mL (comprising 8mol/L urea and 70mmol/L mercaptoethanol), and the denaturant with such high concentration cannot be contained in the vortex renaturation process, so that the protein is subjected to rapid dialysis for 4 hours, the dialyzate is diluted renaturation solution (20 mmol/L Tris-HCl,50mmol/L arginine, 5mmol/L EDTA, 300mmol/L sodium chloride and 0.2mmol/L DTT) before, the dialyzed 1mL concentration of the protein is about 0.2mg/mL, and the dialyzed 1mL concentration is filled into a glass tube with the diameter of 1cm of a vortex renaturation device (figures 6 and 7), and the time for the protein renaturation, the inclination angle and the rotation speed of the glass tube are repeatedly searched, and finally the glass tube angle is 45 degrees, the rotation speed is 5000rpm and the renaturation time is 20min.

Example 2

The CA16 VP1 protein has a coding sequence shown as SEQ ID NO.3, and an amino acid sequence shown as SEQ ID NO. 4; the recombinant vector expressing the protein was constructed, expressed and purified in the same manner as in example 1. The construction result of the CA16 VP1 recombinant expression vector is shown in FIG. 8, and the expression and purification result of the CA16 VP1 protein is shown in FIG. 9. Vortex renaturation after washing and purification of VP1 inclusion body protein is completely the same as in step 7 of example 1.

Example 3

1. Preparation of different immunogens

The denatured and purified EV71 and CA16 VP1 prepared in examples 1 and 2 were taken to have protein concentration of 0.5mg/mL (comprising 8mol/L urea and 70mM mercaptoethanol) and placed in a dialysis bag, the protein solution was placed in the dialysis bag and dialyzed at 4℃for 8h (20 mmol/L Tris-HCl pH8.0,6mol/L urea), and after dialysis for 8h with dialysate (20 mmol/L Tris-HCl pH8.0,4mol/L urea), the dialysate was again changed to (20 mmol/L Tris-HCl pH8.0,2mol/L urea) and dialyzed for 8h with the aid of a diluent (20 mmol/L Tris-HCl pH 8.0) containing no urea, and finally lyophilized after the concentration was measured by the protein BCA method, the immunological dose was 100. Mu.g protein+4.3. Mu.L Al (OH) ₃ adjuvant per mouse as immunogen ①; concentrating the protein solution (6 tubes) subjected to vortex renaturation in the step 6 to about 2mL, dialyzing with 10mmol/L HEPES buffer solution with pH7.0, finally mixing the dialyzed protein with 50 mu L aluminum adjuvant, standing for 10min, and uniformly mixing for several times, wherein the protein concentration is about 1mg/mL by a BCA method, and the immune dose of each mouse is 100 mu g of the protein solution, so that the protein solution is taken as an immunogen ② of the mouse; taking an immune group only added with aluminum hydroxide adjuvant as an adjuvant control group ③; adding formaldehyde with the final concentration of 0.2% into EV71 and CA16 virus liquid, inactivating for 16 hours at 37 ℃, then adding sodium metabisulfite with the final concentration of 0.2% to stop the inactivation, mixing 100 mu L of inactivated virus with the dosage of 10 ⁷ TCID50/ml and 50 mu g of aluminum hydroxide adjuvant for each mouse, and filling up the whole volume to 200 mu L with 10mmol/L of HEPES with pH7.0 as immunogen ④; normal mouse control group ⑤ was additionally added.

2. Immunized mice and antiserum titer detection after mixing of different immunized groups

20 BALB/c mice are selected and randomly divided into 5 groups, 4 of each group, the experimental group comprises VP1 common renaturation and vortex renaturation (100 mug/antigen) in 7, an inactivated virus immune group mixed with aluminum hydroxide adjuvant is used as a positive control group, the rest aluminum hydroxide adjuvant group and a normal mouse group are used as a negative control group, and the mice are subjected to subcutaneous multipoint injection.

(1) First immunization injection:

The prepared antigen and the adjuvant are mixed and then are respectively injected into mice, the injection amount of the antigen of each mouse is 100 mug, the mice are subjected to eyeball blood collection after 14 days of one-time free, the mice are centrifuged at 3000rpm for 10min at 4 ℃, serum is separated, and the mice are frozen at-80 ℃ for ELISA detection (IgG detection).

(2) Second immunization injection: two weeks after one immunization, a second immunization was performed, and the immunization doses were the same. After two weeks of the second immunization, mice were subjected to eyeball blood collection, and the collected blood was centrifuged at 3000rpm for 10min at 4℃to separate serum, and frozen at-80℃for ELISA detection (IgG detection). Mice were subjected to blood collection at the eyeballs three weeks and four weeks after the second immunization, and serum was isolated as above, and frozen at-80℃for ELISA detection (IgG detection).

ELISA antiserum titers detection:

1. sample dilution: samples were diluted with PBS, negative, positive, and blank control were set at the same time.

2. Coating: soluble antigen is taken and added into coating liquid for dissolution, 50 mu l/hole VP1 antigen coating mixed liquid is added, the final concentration is 1 mu g/hole, and the mixture is incubated at 37 ℃ for 2 hours or at 4 ℃ overnight.

3. Washing the plate: and (5) throwing away the liquid, taking a piece of clean gauze, and cleaning the liquid in the plate hole. The plate is washed three times by a plate washing machine, the plate is slapped by force, and the liquid in the plate holes is slapped.

4. Closing: adding 200 μl/well of sealing solution into the wells of the enzyme-labeled plate, incubating at 37deg.C for one hour, and cleaning the plate by beating the sealing solution from the wells on clean gauze after incubation.

5. An antibody: the collected serum was diluted 1:100 and added to the first well of the ELISA plate (100. Mu.l of 1:100 dilution was added to the first well, diluted with blocking solution, followed by 50. Mu.l of blocking solution), followed by dilution at the double ratio and incubation at 37℃for 1.5h.

6. And (2) secondary antibody: (dilution of the secondary antibody to 1:5000-1:8000, dilution with blocking solution) the secondary antibody diluted to working concentration was added to the wells at 50 μl/well and incubated for one hour at 37 ℃.

7. Washing the plate: and (5) throwing away the liquid, taking a piece of clean gauze, and cleaning the liquid in the plate hole. The plate is washed three times by a plate washing machine, and then the plate is beaten by force, so that the liquid in the plate holes is beaten.

8. Color development and reading: TMB color development, adding 50 μl/well of the prepared substrate to the plate, incubating at 25deg.C for 25min, adding 1mol/L HCl 50 μl/well to stop reaction, opening the microplate reader, adjusting to OD ₄₅₀ reading, and recording related data, wherein the negative value is less than 0.06, and the value is greater than 0.2, and the antibody titer is detected as (FIG. 4), wherein the antibody titer is substantially consistent with common dialysis renaturation, and higher than that of the inactivated virus group

3. Detection of neutralizing antibody titres of different immunoadjuvants and VP1 antigen group sera

After ELSIA detection, serum with higher antibody titer is taken for neutralization protection test, and the neutralization protection test comprises the following steps:

1) Dilution of virus: the virus was diluted to 100TCID ₅₀/50 μl in a sterile vessel with DMEM cell culture.

2) Serum dilution: the 6 serums are respectively marked with A, B, C, D, E, F, 0.35ml of cell culture solution containing 10% of diabodies is added into a sterile tube, then 0.05ml of serum sample is added, the mixture is uniformly mixed, the temperature is 56 ℃ and inactivated for 30min, namely 1:8 diluted serum is obtained, and then 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024 diluted serum gradients are prepared by using the cell culture solution containing 1% of diabodies.

3) In 96-well plates, 50 μl of diluted serum was added per well.

4) Mu.l of diluted virus solution at a concentration of 100TCID ₅₀/50. Mu.l was added to each well.

5) Mixing, and incubating in a CO ₂ incubator at 37deg.C for 2 hr.

6) 100. Mu.l of Vero cell suspension in logarithmic growth phase (4-5 passages after resuscitating) was added to the wells, the concentration of the cell suspension was 2X 10 ⁵ cells/ml, and the wells were placed in a CO ₂ incubator at 37℃for incubation.

7) CPE was observed daily using an inverted microscope and recorded, and when the virus control wells were completely diseased, the final results were determined (approximately 5-7 d) and the test results were as shown (fig. 5). The vortex renaturation protein is effectively combined with aluminum adjuvant, and the generated neutralization protection antibody is far higher than that of a common renaturation protein adjuvant group and a control group, and basically can reach 1:64-1:128.

Sequence listing

<110> Beijing Kaiyue medical science and technology Co., ltd

<120> VP1 protein, and preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 888

<212> DNA

<213> EV71 Virus (Human enterovirus 71)

<400> 1

gatcgcgtag ccgacgttat cgaatcttct atcggtgatt ccgtgtctcg cgcgctgact 60

caggctctgc cggcaccgac tggtcagaac acccaggtgt ccagccaccg tctggacacc 120

ggcaaagtgc cggcgctgca ggcagcagaa atcggcgcca gcagcaacgc gtccgacgaa 180

tccatgatcg agactcgctg cgttctgaac tctcactcca ctgctgaaac taccctggat 240

tcctttttca gccgtgctgg cctggttggt gaaatcgatc tgccgctgga aggcaccacc 300

aacccgaacg gctatgcgaa ttgggacatc gacatcactg gctatgcaca gatgcgtcgc 360

aaggtagaac tgtttaccta tatgcgtttc gatgcagaat tcactttcgt tgcttgcacc 420

ccgaccggcc aggtggtacc acagctgctg caatatatgt tcgtgccgcc gggcgcacca 480

aaaccagatt cccgtgaatc tctggcttgg cagactgcga ctaacccgtc tgttttcgta 540

aaactgtccg acccgccggc gcaggtgtct gtgccgttta tgtccccagc ttccgcgtac 600

cagtggttct acgatggtta cccaactttt ggtgagcaca agcaagaaaa agatctggag 660

tatggtgcat gcccgaacaa catgatgggt accttctctg tgcgtaccgt tggtacctcc 720

aaatccaaat acccgctggt agttcgtatc tatatgcgta tgaaacacgt gcgtgcgtgg 780

attccgcgtc cgatgcgtaa ccagaactac ctgttcaaag ccaacccgaa ctacgcaggt 840

aacagcatca aaccgaccgg cgcttcccgt gcggcgatca ctaccctg 888

<210> 2

<211> 458

<212> PRT

<213> EV71 Virus (Human enterovirus 71)

<400> 2

Met Ser Asp Lys Ile Ile His Leu Ser Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser Ile Gly Asp Ser

165 170 175

Val Ser Arg Ala Leu Thr Gln Ala Leu Pro Ala Pro Thr Gly Gln Asn

180 185 190

Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys Val Pro Ala Leu

195 200 205

Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser Asp Glu Ser Met

210 215 220

Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr Ala Glu Thr Thr

225 230 235 240

Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val Gly Glu Ile Asp Leu

245 250 255

Pro Leu Glu Gly Thr Thr Asn Pro Asn Gly Tyr Ala Asn Trp Asp Ile

260 265 270

Asp Ile Thr Gly Tyr Ala Gln Met Arg Arg Lys Val Glu Leu Phe Thr

275 280 285

Tyr Met Arg Phe Asp Ala Glu Phe Thr Phe Val Ala Cys Thr Pro Thr

290 295 300

Gly Gln Val Val Pro Gln Leu Leu Gln Tyr Met Phe Val Pro Pro Gly

305 310 315 320

Ala Pro Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr

325 330 335

Asn Pro Ser Val Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser

340 345 350

Val Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly

355 360 365

Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly

370 375 380

Ala Cys Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly

385 390 395 400

Thr Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met

405 410 415

Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn Tyr

420 425 430

Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys Pro Thr

435 440 445

Gly Ala Ser Arg Ala Ala Ile Thr Thr Leu

450 455

<210> 4

<211> 891

<212> DNA

<213> Coxsackie virus A16 type (Coxsachie virus)

<400> 4

ggtgatccga ttgctgatat gattgaccaa accgtaaaca accaagttaa ccgctctctg 60

actgcgctgc aagttctgcc aactgctgcg aataccgagg cgtcctccca ccgcctgggc 120

actggcgttg ttccagctct gcaagctgct gagactggtg cgtctagcaa cgcctccgac 180

aagaacctga ttgagactcg ctgtgttctg aaccatcaca gcacccagga aaccgccatc 240

ggcaacttct tctcccgtgc tggcctggta tctatcatta ccatgccgac tactggcacc 300

cagaataccg acggctacgt aaactgggat attgatctga tgggctacgc gcaactgcgt 360

cgtaagtgcg agctgttcac ttacatgcgt ttcgatgctg agtttacctt tgtggtagcc 420

aaaccgaacg gcgaactggt tccgcaactg ctgcaataca tgtacgtacc accgggtgca 480

ccgaaaccga cctctcgcga tagctttgcc tggcagactg caaccaatcc gtccgtgttc 540

gttaagatga ccgacccacc ggctcaggta tccgttccgt tcatgtctcc ggcatccgcg 600

taccagtggt tctatgacgg ctacccgacc ttcggcgagc atctgcaagc aaacgacctg 660

gactacggtc agtgcccaaa caacatgatg ggtactttca gcatccgtac tgtgggcact 720

gagaaatctc cgcactccat cactctgcgt gtttacatgc gcatcaaaca cgttcgtgcg 780

tggattccgc gtccactgcg taaccagcca tacctgttta agactaaccc gaactacaaa 840

ggtaacgaca tcaaatgcac ctctacctcc cgtgataaga tcaccaccct g 891

<210> 4

<211> 297

<212> PRT

<213> Coxsackie virus A16 type (Coxsachie virus)

<400> 4

Gly Asp Pro Ile Ala Asp Met Ile Asp Gln Thr Val Asn Asn Gln Val

1 5 10 15

Asn Arg Ser Leu Thr Ala Leu Gln Val Leu Pro Thr Ala Ala Asn Thr

20 25 30

Glu Ala Ser Ser His Arg Leu Gly Thr Gly Val Val Pro Ala Leu Gln

35 40 45

Ala Ala Glu Thr Gly Ala Ser Ser Asn Ala Ser Asp Lys Asn Leu Ile

50 55 60

Glu Thr Arg Cys Val Leu Asn His His Ser Thr Gln Glu Thr Ala Ile

65 70 75 80

Gly Asn Phe Phe Ser Arg Ala Gly Leu Val Ser Ile Ile Thr Met Pro

85 90 95

Thr Thr Gly Thr Gln Asn Thr Asp Gly Tyr Val Asn Trp Asp Ile Asp

100 105 110

Leu Met Gly Tyr Ala Gln Leu Arg Arg Lys Cys Glu Leu Phe Thr Tyr

115 120 125

Met Arg Phe Asp Ala Glu Phe Thr Phe Val Val Ala Lys Pro Asn Gly

130 135 140

Glu Leu Val Pro Gln Leu Leu Gln Tyr Met Tyr Val Pro Pro Gly Ala

145 150 155 160

Pro Lys Pro Thr Ser Arg Asp Ser Phe Ala Trp Gln Thr Ala Thr Asn

165 170 175

Pro Ser Val Phe Val Lys Met Thr Asp Pro Pro Ala Gln Val Ser Val

180 185 190

Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr

195 200 205

Pro Thr Phe Gly Glu His Leu Gln Ala Asn Asp Leu Asp Tyr Gly Gln

210 215 220

Cys Pro Asn Asn Met Met Gly Thr Phe Ser Ile Arg Thr Val Gly Thr

225 230 235 240

Glu Lys Ser Pro His Ser Ile Thr Leu Arg Val Tyr Met Arg Ile Lys

245 250 255

His Val Arg Ala Trp Ile Pro Arg Pro Leu Arg Asn Gln Pro Tyr Leu

260 265 270

Phe Lys Thr Asn Pro Asn Tyr Lys Gly Asn Asp Ile Lys Cys Thr Ser

275 280 285

Thr Ser Arg Asp Lys Ile Thr Thr Leu

290 295

Claims

1. A preparation method of VP1 protein is characterized in that VP1 inclusion body protein induced to be expressed is subjected to washing, urea dissolution, denaturation purification and renaturation to obtain VP1 protein; the renaturation adopts an eddy renaturation device to carry out eddy renaturation, the inner diameter of a glass tube of the eddy renaturation device is 1cm, the length of the glass tube is 16cm, the angle of the renaturation glass tube is 30-80 degrees, the rotating speed is 3000-8000rpm, the renaturation time is 10-30min, and the VP1 protein is selected from VP1 protein of EV71 or CA 16.

2. The method according to claim 1, wherein the washing is performed by washing the obtained VP1 inclusion body protein with a washing buffer 1, a washing buffer 2 and a washing buffer 3 in sequence, and finally rinsing with ultrapure water, wherein the obtained protein is the washed inclusion body protein; wherein,

3. The preparation method of claim 1, wherein the urea dissolution is to dissolve the washed inclusion body protein with a high-concentration urea solution, and the supernatant obtained by centrifugation is the inclusion body protein after urea dissolution, and the high-concentration urea solution has a formula of 20mmol/L Tris-HCl pH8.0,8mol/L urea and 70mmol/L beta-mercaptoethanol.

4. The method according to claim 1, wherein the denaturation and purification are performed by subjecting the inclusion body protein to strong anion exchange purification and Sepharose CL-4B gel filtration chromatography in this order.

5. The preparation method of claim 4, wherein the strong anion exchange purification is carried out by using a strong anion exchange column filler which is pre-filled with acid-base rinsed to carry out strong anion exchange purification on protein after inclusion body dissolution, preferably washing the column with ultrapure water, then using an equilibrium buffer until a baseline is balanced, slowly loading a VP1 protein sample, washing the column with the equilibrium buffer again until the baseline is balanced, eluting the balanced sample with an elution buffer, and purifying to obtain target protein, wherein the formulation of the equilibrium buffer is 20mmol/L Tris-HCl pH8.0,8mol/L urea and 70mmol/L beta-mercaptoethanol; the elution buffer solution formula is 20mmol/L Tris-HCl pH8.0,8mol/L urea, 70mmol/L beta-mercaptoethanol, 80mmol/LnaCl; the method for the gel filtration chromatography of the Sepharose CL-4B comprises the steps of carrying out PEG20000 dialysis bag embedding concentration on a VP1 protein sample subjected to strong anion exchange purification, carrying out the gel filtration chromatography purification analysis of the concentrated sample, wherein the concentration of the concentrated sample is 1.0-1.5 mg/ml.

6. The method according to claim 1, wherein the purified denatured protein is subjected to rapid dialysis for 4 hours before renaturation, wherein the dialysate is 20 mmol/L Tris-HCl,50 mmol/L arginine, 5 mmol/L EDTA,300 mmol/L sodium chloride, 0.2 mmol/L DTT, and the dialyzed protein solution is 1mL at a concentration of 0.1-0.5 and is packed into a glass tube having a length of 16cm and a diameter of 1cm for vortex renaturation, wherein the glass tube has an angle of 30-60 °, a rotation speed of 3000-6000rpm, and a renaturation time of 10-40min.

7. The method according to claim 1, wherein the vortex renatured protein solution is concentrated and dialyzed against 5-20mmol/L HEPES buffer pH 7.0.