CN105853998B - Osteopetrosis correlation transmembrane protein is treating or preventing the application in EV71 infection medicine - Google Patents

Abstract

The present invention discloses the application of osteosclerosis-associated transmembrane protein in treating or preventing EV71 infection. The steps are as follows: first express Ostm1 protein in insect cells (SF9) using insect baculovirus expression system, and then use nickel column affinity Ostm1 protein was purified by chromatography, and finally the antiviral effect of Ostm1 protein was verified in malignant embryonic rhabdomyoma cells (RD cells) infected with EV71. The experimental results prove that the purified Ostm1 protein can inhibit the replication of EV71 virus at the mRNA level and protein level at a certain concentration. The invention evaluates the anti-EV71 virus effect of the osteosclerosis-associated transmembrane protein (Ostm1) from the aspects of protein purification and cell nucleus molecules, and lays the foundation for its further development and application. The protein can directly inhibit the replication of EV71 virus at an appropriate concentration, and the effect is obvious, there is no side effect on cells, and the use is simple and convenient. It shows that the drug has the prospect of being developed as an effective drug against EV71 virus and applied in clinic.

Description

Application of osteosclerosis-associated transmembrane protein in the treatment or prevention of EV71 infection

technical field

The present invention relates to the field of antiviral drugs, and relates to a new application of an osteopetrosis associated transmembrane protein (Osteopetrosis associated transmembrane protein 1), and more specifically relates to the application of an osteopetrosis associated transmembrane protein in the treatment or prevention of EV71 infection .

Background technique

Human enterovirus 71 (Hu-man enterovirus) belongs to the genus Enterovirus of the family Picomaradae. The EV71 virus particle is an icosahedral symmetrical spherical structure without envelope and spikes, and its diameter is about 24-30nm. The full length of the viral genome is 7408bp, which is a single-stranded positive-sense RNA. There is only one open reading frame (ORF) in the genome, with 5' and 3' non-coding regions (UTRs) on both sides of the ORF, and at the end of the 3' end of the genome There is also a polyadenylic acid tail (poly-A) of variable length. The ORF in the viral genome can encode 2194 amino acids, and the polyprotein composed of these amino acids can produce a total of three precursor proteins, P1, P2 and P3. Among them, the P1 precursor protein encodes 4 viral coat proteins, namely VP1, VP2, VP3 and VP4; the P2 and P3 precursor proteins encode a total of 7 non-structural proteins. Among them, the VP1 protein is the most important capsid protein, which contains the main antigenic determinant of EV71. The gene sequence encoded by it has a high correlation with the serology of the virus, so it is often used as the identification and gene expression of the virus type. evolutionary analysis. The capsid of the virus particle is composed of 60 subunits, and each subunit has a pentamer-like structure formed by VP1-VP4, and its antigenic determinants are basically located on VP1-VP3.

The EV71 virus invades the upper respiratory tract, throat and intestinal tract of the human body. It first collects and initially amplifies in the local mucosa, tonsils, pharynx and other lymphoid tissues and intestinal lymph nodes, and then releases the EV71 virus into the blood, forming the first In secondary viremia, the EV71 virus spreads with the blood to the target tissue with its receptors, and multiplies again to cause the second viremia and some clinical symptoms. Infection with EV71 can cause long-term sequelae, such as neurological sequelae and delayed and reduced cognitive function. Pulmonary edema and hemorrhage are the main causes of death in children infected with EV71.

At present, there is no effective antiviral drug to treat severe EV71 infection, and there is no vaccine that has entered the clinical stage. Therefore, avoiding contact with infected patients and suspected infected patients is the main method to prevent EV71 infection. In the research of anti-EV71 virus drugs, pyridyl imidazolinone is a new type of adhesive drug, which can interact with the VP1 protein of EV71, and interfere with VP1 and its interaction by expanding the hydrophobic region of the VP1 gene. Receptor-binding approach to inhibit EV71 replication. In addition, cytokine-mediated methods can be used to treat edema caused by EV71 infection, for example, ribavirin can reduce the mortality rate of EV71 infection and subsequent paralysis sequelae by reducing the viral load in infected mouse tissues . Finally, we can prevent EV71 infection by vaccination. Current EV71 vaccine candidates mainly include live attenuated vaccines, genetically engineered vaccines, DNA vaccines, virus-like particles, and genetically modified vaccines. These vaccines have been evaluated in animal experiments. But clinical trials have yet to take place.

The osteopetrosis-associated transmembrane protein (Ostm1) has 338 amino acids and has a single transmembrane domain, a short cytoplasmic tail, a long luminal domain containing multiple hyperglycosylation sites and A signal peptide that can be cleaved. The luminal domain of the Ostm1 protein contains a weak RING-finger protein. The original function of this protein may be similar to E3 ubiquitin ligase.

Ostm1 protein can be expressed in osteoclasts, hematopoietic stem cells and other hematopoietic cell families such as B cells, NK cells and mast cells. Ostm1 gene can play an important role in the differentiation of hematopoietic stem cells independently of CIC-7 gene. In addition, Ostm1 protein can also regulate the typical Wnt signaling pathway, thus playing an important role in bone homeostasis. Mutations in the Ostm1 gene in patients with osteopetrosis produce a truncated protein that lacks a transmembrane domain and a cytoplasmic tail that allows the protein to be secreted, inhibits Wnt signaling and binds Inhibitor of osteocyte precursors thereby inhibiting osteoclast production. Ostm1 protein is the target site of miR-140 in pluripotent stem cells, which has anti-adipogenic function, and Ostm1 protein also has the potential to connect bone morphogenetic protein (BMP) and Wnt signaling pathway. However, there is no literature report on the relationship between Ostm1 protein and viral diseases and related viruses, and there is no report on the preventive and therapeutic effects of this virus on the large-scale epidemic and relatively highly pathogenic enterovirus (EV71) .

Contents of the invention

The purpose of the present invention is to provide the application of a human osteosclerosis-associated transmembrane protein in the preparation of medicines for the treatment and prevention of human enterovirus EV71 infection. The function of EV71 virus was evaluated, which laid a good foundation for its further development and application. The protein can directly inhibit the replication of EV71 virus at an appropriate concentration, and the effect is obvious. It has no side effects on cells and is easy to use.

In order to achieve the above-mentioned purpose, the present invention realizes through the following technical solutions:

The application of an osteosclerosis-associated transmembrane protein in the preparation of a drug for treating or preventing enterovirus 71 (EV71) infection, the steps of which are:

(1) Connect the C-terminus of the Ostm1 gene (GenBank NP054747) with a His tag, and then clone it into the PH promoter (on the plasmid) of the pFastBac Dual vector (Invitrogen), and obtain the correct clone pFastBac Dual-Ostm1 by sequencing ;

(2) Transform pFastBac Dual-Ostm1 into Escherichia coli (DH10β) (purchased from Crowning Biotechnology Co., Ltd.), and obtain the baculovirus Bacmid-Ostm1 carrying the Ostm1 gene through Tn7 transposon (DH10β) gene recombination;

(3) Bacmid-Ostm1 was transfected into SF9 cells (from China Center for Type Culture Collection) to obtain P1-generation recombinant virus, and the most active P3-generation recombinant baculovirus was obtained after virus amplification, and the successful expression of Ostm1 protein was proved ;

(4) SF9 suspension cells were infected with P3 virus for large-scale protein expression, and then the purified Ostm1 protein was successfully obtained by nickel column affinity chromatography;

(5) The purified Ostm1 protein was applied to RD cells infected with EV71 virus (GenBankJN230523.1) at different concentrations, the total RNA was extracted after 8 hours, and the VP1 structural protein of the virus was used as a marker to detect the replication of EV71 virus, respectively. The antiviral effect of the protein was detected at the RNA and protein levels.

The above experimental results show that the purified Ostm1 protein can significantly inhibit the mRNA level of EV71 virus VP1 at a certain concentration, and the Western blot results also show that the protein can inhibit the expression of VP1 protein level at the same concentration. Thus, it is proved that the protein has the function of anti-E71 virus replication.

The present invention discovers the new medical value of the transmembrane protein associated with osteosclerosis, and provides new and effective drugs for resisting enterovirus type 71, treating and/or preventing EV71 infection.

The application of the described a kind of osteosclerosis-associated transmembrane protein in the preparation of medicine for treating or preventing enterovirus 71 (EV71) infection, the specific application is to directly act on the cells infected with EV71 with the purified Ostm1 protein at an appropriate concentration , the protein can significantly inhibit the replication of EV71 (as shown in Figure 5)

At present, there is no effective antiviral drug to treat severe EV71 infection, and there is no vaccine that has entered the clinical stage. Therefore, avoiding contact with infected patients and suspected infected patients is the main method to prevent EV71 infection. In the research of anti-EV71 virus drugs, pyridyl imidazolinone is a new type of adhesive drug, which can interact with the VP1 protein of EV71, and interfere with VP1 and its interaction by expanding the hydrophobic region of the VP1 gene. Receptor-binding approach to inhibit EV71 replication. In addition, cytokine-mediated methods can be used to treat edema caused by EV71 infection, for example, ribavirin can reduce the mortality rate of EV71 infection and subsequent paralysis sequelae by reducing the viral load in infected mouse tissues . Finally, applicants can prevent EV71 infection through vaccination. Currently, candidate vaccines for EV71 mainly include attenuated active vaccines, genetic engineering vaccines, DNA vaccines, virus-like particles, and genetically modified vaccines. These vaccines have been obtained in animal experiments. Evaluation but clinical trials have not yet been conducted.

Compared with the prior art, the present invention has the following advantages and effects:

The invention evaluates the anti-EV71 virus effect of the osteosclerosis-associated transmembrane protein (Ostm1) from the aspects of protein purification and cell and molecule, and lays the foundation for its further development and application. It shows that the drug has the prospect of being developed as an effective drug against EV71 virus and applied in clinic.

Finally, through experiments, the applicant found that the transmembrane protein associated with osteopetrosis can significantly inhibit the replication of EV71 virus at a concentration of 13.5ng/ml, which suggests that the applicant can make the protein into a related drug, so as to It exerts its antiviral effect during the prevention and post-infection treatment of EV71 virus.

Description of drawings:

Fig. 1 is a schematic diagram of the time and expression environment identification of a P3 generation baculovirus expressing Ostm1 protein.

The results proved that the expression of OStm1 protein was the best on the third day after P3 virus infected insect cells (sf9), and the protein was expressed intracellularly.

Fig. 2 is a gradient elution diagram of different imidazole concentration for protein purification by nickel column affinity chromatography.

The results proved that when the imidazole concentration was 112mM, the Ostm1 protein could be eluted in large quantities, and there were few foreign proteins, so that the purified protein could be obtained.

3 and 4 are schematic diagrams showing that Ostm1 protein at different concentrations inhibits the mRNA level of EV71 virus.

The result shows that when the Ostm1 protein concentration is 13.5ng/ml and 337.5ng/ml, the mRNA level of the VP1 protein of the EV71 virus has a significant decline compared with the control group without virus, indicating that when the protein is at this concentration, the EV71 virus The production of mRNA has a more obvious inhibitory effect.

Fig. 5 is a schematic diagram showing that different concentrations of Ostm1 protein inhibit the level of EV71 virus protein.

The results show that when the protein concentration is 189ng/ml and 360ng/ml, compared with the control group that only adds virus, the protein level of VP1 has a significant decline, indicating that at this concentration, Ostm1 protein has a better effect on the replication of EV71 virus. Obvious inhibitory effect.

Detailed ways

Example 1:

The application of a transmembrane protein associated with osteopetrosis in the preparation of a drug for preventing and/or treating EV71 infection, the steps of which are:

1. Expression and purification of Ostm1 protein in insect baculovirus expression system:

First, the applicant used the cDNA of HepG2 cells (from the China Center for Type Culture Collection) as a template, used specific primers (sequence as follows) to amplify the target gene, and then connected it to the plasmid vector pCAGG-HA (Invitrogen) . The primer sequences are as follows:

Forward: GCGACCGAATTCATGGAGCCGGGCCCGACAGC

Reverse: CGCGCTCGAGGTTTGAATTTTCCTGAATATTGC

Then use specific primers to amplify the target gene from the above plasmid, and clone it into the pFastBacDual vector. The sequences of the primers are as follows:

Forward: CTGAATTCATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTTATGGTCGTGTACATT

TGGTCGTGTACATTTCTTACATCTATGCGATCGAGGGAAGGGAGCCGGGCCCGACAG

Reverse: CGCTCGAGTTAGTGATGGTGATGGTGATGCCTTCCCTCGATTCAGTTTGAATTTTC

Then transform the pFastBac Dual-Ostm1 plasmid into Escherichia coli competent cell DH10β, spread it on the LB solid plate added with kanamycin, tetracycline, gentamycin, x-gal and IPTG, and then put it on Incubate in a 37°C constant temperature biochemical incubator in the dark for 72 hours. Then pick 3 white colonies, re-stretch and culture them on the above-mentioned solid plate, pick a single clone colony after 72 hours, inoculate it into the LB liquid medium containing kanamycin, tetracycline and gentamycin, 200rpm, Incubate overnight at 37°C with shaking. Plasmids were extracted the next day, and the insertion of the Ostm1 gene was identified using the M13 primer. M13 primer sequence is as follows

Forward: GTAAAACGACGGCCAGT

Reverse: AACAGCTATGACCATG

Take out the frozen SF9 cells (from the China Center for Type Culture Collection) from the liquid nitrogen tank, put them into a 37°C water bath quickly, thaw quickly, and recover the cells. Then add 4ml of SF900 The culture was based on T25 cell flasks and cultured in a 27°C carbon dioxide-free cell culture incubator. When the cells can be passaged well, transfect the obtained shuttle plasmid Bacmid-Ostm1 (see 2 in the technical scheme) into SF9 cells, and after 72 hours of transfection or when the cells appear lesions, collect the culture supernatant and transfer to the Bacteria in a 15ml centrifuge tube, centrifuge at 1000rpm for 10min to obtain the P1 generation baculovirus, and then continue to infect the SF9 cells in a good growth state to obtain the P2 generation baculovirus, and in this way to obtain the P3 generation baculovirus If the virus is stored for a long time, the virus should be stored at -80°C.

In order to verify the expression of Ostm1 protein in SF9 cells, the applicant took the P1 generation, P2 generation, P3 generation and P3 generation virus-infected SF9 cells 1d, 2d, 3d cells and culture supernatants for Western-blot experiments. Through the experimental results, the applicant obtained that the P3 generation virus had the best expression of Ostm1 protein on the third day of infection, and the protein was expressed in a non-secretory form.

After confirming the successful expression of the Ostm1 protein, the next step is to purify the protein. The first is the cultivation of SF9 suspension cells: take out the frozen SF9 suspension cells from the liquid nitrogen tank, and inoculate them into sterile shake flasks (generally, the inoculum volume is 1/3 of the shake flask volume). 110rpm, shake culture at 27°C; observe and measure the number and survival rate of cells every day, when the concentration of cells reaches ⁴ ×106/ml, and the survival rate is above 95%, the cells can be passaged; Add an appropriate amount of fresh culture medium to the original culture flask to make the cell concentration reach 2×10 ⁶ cells/ml, and then divide it into two sterile shake flasks. Continue shaking at 110 rpm at 27°C. When the density of SF9 suspension cells reaches 2×10 ⁶ cells/ml, add P3 generation virus with MOI=1, continue culturing for 3 days, discard the medium, and collect the cells. Weigh the wet weight of insect cells, and then add insect cell lysate at a ratio of 1:10. Place the cells under the sonicator, and set the time and frequency of sonication (generally 2s for 2s, 30min for about 30min). During the whole sonication process, the cells need to be kept on the ice box, and the supernatant is collected after the sonication is over. Here, the applicant uses the BioLogic DuoFlow protein purification instrument of BIO-RAD Company to purify the protein. The specific experimental steps are (1) wash the nickel column with deionized water: A pump (included in the above machine) and Put B (included in the above machine) into water, flow rate 1.5ml/s, and wash for 20 minutes; (2) Balance nickel column: A pump is put into the balance buffer, B pump is put into water, and only A pump works in this step , the flow rate is 1.5ml/s, and the balance is 20min; (3) Sample loading: put the A pump into the sample, and the B pump into the water. In this step, only the A pump works, the flow rate is 1.5ml/s, and the time is determined by the sample volume. Part of the effluent; (4) Rinsing: Put the A pump into the rinsing buffer, and put the B pump into the water. In this step, only the A pump works, the flow rate is 1.5ml/s, rinse for 20min, and collect the rinsing solution; (5) Linear gradient Elution: Put the A pump into high concentration imidazole (200mM), put the B pump into water, the two pumps work at the same time in this step, set the number of tubes for collection. The concentration of the eluent changes linearly from 0-200mM, and each eluate is collected; (6) High-concentration rinsing: put A pump into high-concentration imidazole (200mM), and B pump into water. This step only has A The pump is working, the flow rate is 2ml/s, and rinsed for 15min to wash away the remaining protein on the nickel column; (7) Cleaning system: Put the A pump and the B pump into 20% ethanol at the same time, and the flow rate is 2ml/s , wash for 30min. Make the whole system in 20% ethanol environment, and finally save the two pumps in 20% ethanol solution.

Note: A pump must be cleaned with deionized water during the replacement process.

2. Anti-EV71 virus activity research of purified Ostm1 protein:

2.1 Cells and viruses:

The human rhabdomyosarcoma cell line RD was obtained from the China Type Culture Collection (CTCC) (Wuhan University) and preserved by our laboratory.

The EV71 virus strain was isolated by our laboratory.

2.2 Experimental method:

Spread the RD cells in the logarithmic phase on a 12-well plate. When the RD cells adhere to the wall and the cells basically cover each well, replace with serum-free medium, and then only leave one well as a control without adding virus. EV71 virus was added to the wells at MOI=1. After 90 minutes, the original medium was discarded, and then fresh serum-free medium was added, and purified Ostm1 protein of different quality was added to each well, and the cells were harvested after 8 hours, and the total RNA in the cells was extracted by the Trizol method, reverse-transcribed Become cDNA, and then use the method of fluorescent quantitative PCR to detect the change of VP1 protein in the EV71 virus at the RNA level. The primer sequences are as follows:

VP1: Forward AATTGAGTTCCATAGGTG

Reverse CTGTGCGAATTAAGGACAG

GAPDH: Forward AAGGCTGTGGGCAAGG

Reverse TGGAGGAGTGGGTGTCG

Because the optimal concentration range of Ostm1 protein to inhibit virus replication is not known, the applicant enlarged the mass range (6.75ng-1350ng) of Ostm1 protein added to cells in previous experiments to screen its most suitable concentration. The experimental results are shown in Figure 3.

It can be seen from Figure 3 that when the amount of Ostm1 protein added was 6.75ng, 33.75ng and 270ng, the mRNA level of VP1 protein decreased significantly compared with the control group that only added virus, indicating that Ostm1 protein had an effect on the mRNA of EV71 virus at this concentration. The production has a more obvious inhibitory effect. However, when the concentration of Ostm1 protein was continuously increased, it was found that it had no inhibitory effect on the replication of EV71 virus. In order to further verify the above experimental results, in the next experiment, the applicant further narrowed the mass range of the protein added to the cells (13.5ng-405ng), and the experimental results are shown in Figure 4:

As can be seen from Figure 4, when the quality of the Ostm1 protein added is 13.5ng and 337.5ng, the mRNA level of the VP1 protein of the EV71 virus is significantly lower than that of the control group without the virus, indicating that when the protein is at this concentration, it is on EV71. The production of viral mRNA has a more obvious inhibitory effect. This is basically consistent with the experimental results in Figure 4, from which the applicant can conclude that the Ostm1 protein can inhibit the production of EV71 virus mRNA in an appropriate concentration range.

The RD cells in the logarithmic phase were plated on a six-well plate, and the cells were completely attached to the wall and covered the six-well plate. Discard the original medium and replace with serum-free medium. One of the wells was used as a control without adding virus, and EV71 virus was added to each well at MOI=1. After 90 min, the original medium was discarded and replaced with fresh serum-free medium. Different masses of Ostm1 protein were added to each well. After 12 h, the cells were collected and disrupted by ultrasonic waves. Then Western-blot experiment was used to detect the change of VP1 protein in EV71 virus under different protein concentrations. The experimental results are shown in Figure 5:

In Fig. 5, the mass of the added Ostm1 protein was 7ng, 21ng, 63ng, 189ng, 360ng and 567ng in sequence. It can be seen from Figure 5 that when the added protein was 189ng and 360ng, the protein level of VP1 decreased significantly compared with the control group only adding virus, indicating that at this concentration, the Ostm1 protein has a significant effect on the replication of EV71 virus. inhibitory effect.

Through the double verification at the mRNA level and the protein level, it is proved that the Ostm1 protein does inhibit the replication of the EV71 virus within a certain concentration range, which also proves that the Ostm1 protein expressed and purified by the baculovirus expression system has Active.

Claims (1)

1. The application of the osteosclerosis-associated transmembrane protein Ostml whose sequence is GenBank NP054747 in the preparation of medicines for treating or preventing EV71 infection.