TW201100545A - Method for preparing enterovirus virus-like particle and use thereof - Google Patents

Abstract

The present invention is related to a method for preparing enterovirus 71 virus-like particle in the baculovirus/insect cell expression system and use thereof. By modifying the baculovirus vector design, the present invention lowers the expression level of protease 3CD and increases the expression level of the capsid protein precursor P1 so as to increase VLP yield. These VLPs can elicit high antibody titers and neutralization titers responses in mice, demonstrating to be as an enterovirus 71 vaccine.

Description

201100545 VI. Description of the Invention: [Technical Field] The present invention relates to a method for producing an enterovirus 71 type virus particle by using a baculovirus recombinant protein expression system, a method for producing the same, and an application thereof. [Prior Art] Enterovirus is classified as Picomaviridae 'p0Sitive, singie strand RNA virus' infection is mainly transmitted through feces or respiratory droplets, which can infect various systems of the human body. Organs, even the phenomenon of organ failure. Traditionally, neutralizing serum tests can be used to divide enterovirus into poliovirus type (p〇li〇vjmses, PV), Croxackieviruses A (CAV), and kesaki virus type B ( Coxsackieviruses 2B, CBV) 'Enteric cytopathogenic human orphan viruses' (Ek〇viruses), and enterovirus type 68_71. Due to advances in gene sequencing methods in recent years, enterovirus classification has been subdivided into four types of A_D, and there are currently more than one hundred ash-clear types. Enterovirus 71 shirt _ 丨 71 71, EV7i) 疋 刖 has found at least one of the serotypes of enterovirus. According to the differences in the sequencing of their genetic molecules, they can be divided into three groups: A, B, and C, which can be subdivided into A bi b5.

The different subtypes of Cl C5 ' are related to the evolution and regionality of their viral genes. In the serotypes of enteroviruses, the virulence of EV71 is particularly high, often accompanied by complications of the nervous system and the possibility of death. 201100545 Enterovirus 71 virus particles are icosahedral polymeric macromolecules with a diameter of about 7 nm, but the detailed group scales have not been clearly studied. At present, the resident of the enterovirus comes from the ridge to listen to gray matter and sputum. _, silkworm virus research, enterovirus genome length of about 7500nt 'including single-open reading frame _ fr継, ORF about 6600m), on both sides are 5, and 3, untranslated area (she muscle age ^ Mail (10), UTR). This ORF can be divided into three regions: decay, p2 and p3, and will be translated into a single polyprotein (P〇lypr〇tein). P1 carries viral capsid proteins: VP4, W2, VP3 and . P2 and p3 carry non-structural proteins such as pr〇tease 2A, 3C, and 3CD, as well as virally polymerized plum 3DP', which is primarily responsible for viral replication and virus toxicity. Taking pediatric pruritus virus as an example, enterovirus granules are theoretically formed by 6Q Cong and Vm59, and a W0 molecule (VP2 and a precursor before unseparation). VP VP2 and VP3 are located on the outer layer of the scale, while the side is completely located on the inner layer. Many of the enteroviruses that have been studied, such as poliovirus, CAV and CBV, have been found to have many neutralizing epitopes (mainization determinants) found mainly in VP1 and a few in VP2 and VP3. In addition, the self-quality of these structural eggs is also related to the weakening of viral toxicity, thermal stability of the virus, host range, and cell driveability. Baculovims, generally referred to as baculoviruses, belong to the baculoviride family, whose host is confined to invertebrates. It is reported that more than 6 species of insects have been infected. Lepidoptera is its main host. According to the latest classification of the International Committee on Taxonomy of Virus in 2004, the baculoviridae can be divided into two genera, the genus genus 201100545 (Gnmulovims, GW human anti-polar corpus callosum Nuck) 〇p〇lyhedr〇virus, delete) Composition, the difference lies in the difference in embedded protein. The former has a small embedding body of about 0.25-0.5 μm, and only one virion is embedded; the latter is larger, approximately, consisting mainly of polyhedrin (poiyhecjron) having a size of about 29 kD, which can be coated. More virions. NPV is cultivated in stable cell strains with its Na-Zhong Zhoucheng gene formula, which has the potential to develop into a gene expression vector. At present, the virus most commonly used as a baculovirus expression vector is Aut〇graph cah: f〇mica O nuclear Polyhedrosis Vi (10); AcMNPV and Bombyx mori nuclear polyhedrosis virus (and Chaga midear polyhedrosis virus; BmNPV). ) 'The main hosts are moths and silkworms. The genetic material of NPV is a double-stranded circular structure of about 8〇2〇〇kb, which is covered by a long-chain nucleocapsid, and the outer layer is made up of a lipoprotein envelope (Ηρ〇_^ envelope). The coating forms a viral structure of about 4 〇 5 〇 nm and a width of about 2 〇〇 4 〇〇 nm. The virus can produce two types of virions after infection: the budding virions O^ded virion; BV) and the embedded virions (〇cdusi〇n b〇dy_derived 〇V). The budding virions are produced in the early stage of the infection cycle. After infection, the new nucleocapsid will be released from the nuclear membrane into the cytoplasm and further penetrate the cell membrane; the embedded virions will be produced in the later stage of the infection cycle, and they will be packaged. Buried in a protein f that is about the size of a polyhedral protein or crystal structure, a polyhedron of about size is formed or called an embedding body. The baculovirus commonly used in laboratories is a budding virus particle. 201100545 The performance of baculovirus is based on the improvement and development of baculovirus. After more than 20 years of improvement and development, it has been widely used in the expression system of heterologous genes, and it is a system for rapid and convenient production of exogenous recombinant proteins. Thus, more than hundreds of viruses, cells, fungi, protozoa, and animal and plant genes have been used to produce recombinant protein f using this system. Even some of the more complex protein structures, such as virions, can be expressed in the host. The baculovirus expression vector system is a kind of eukaryotic cell, and it is expected that the gene is ligated into the baculovirus downstream of the promoter (pr_(4), and then the recombinant baculovirus is used to infect the insect cells. The viral gene promoter may drive this-foreign gene to produce a recombinant protein. Since the insect cells infected by the recombinant baculovirus are eukaryotic cells, the white matter can be modified to various post-translational CpostA-odifieations. Baculovirus Expression _ The eukaryotic gene recombinant protein produced by the system is usually similar in antigenicity, immunity and biological activity to its natural protein. In addition, the promoter used in general is the strong promoter of the baculovirus gene itself (such as P〇1yhedrin or _promoter), so the recombinant protein produced by this system is quite large (1-600mg). /L). In addition to the excellent _ secret correction, the baculovirus secret system has the following advantages. (1) The cashew virus has a considerable genome (88 200kbp), which can accommodate a considerable foreign gene 'making the same strain Baculovirus can carry more than two sets of exogenous genomes simultaneously. (2) Because of recombination; the pathogen of the virus is confined to invertebrates, and it is safer than other gene vectors such as adeno-associated virus (AAV) in 201100545. (3) The recombinant baculovirus itself has sufficient genetic information to infect cells and multiply, so there is no need for the presence of helper cell lines or helper viruses, which is more extensive in subsequent applications. For example, the production of adeno-associated virus (AAV) often requires the presence of an adenovirus, causing some bottlenecks in the purification of AAV. The recombinant baculovirus/insect cell system has the characteristics of suspension culture and can be produced on a large scale by using a reactor, which is more convenient and rapid for subsequent amplification in the production process. ❽ Today's enterovirus 71 vaccines are mainly developed in the following ways: (1) The virus is deactivated by fumarin to produce a deactivated virus vaccine. (2) The laboratory of Professor He Meixiang of the Academia Sinica used multiple virus subcultures and screenings to select new strains with strong fertility and weak toxicity, and hoped to use this new strain as attenuated. Attenuated vaccine» This attenuated virus vaccine has had good results in preliminary mouse animal immunoassay experiments. 〇 (3) Using genetic mutations to produce EV71 strains with reduced toxicity as immune antigens. The resulting immune sera have been found to have the ability to neutralize different serotype viruses in monkey immunization experiments, and the neurotoxicity induced by the mutated EV71 attenuated virus is rather mild. (4) Prof. He Meixiang from the Academia Sinica and Professor Shi Xinru from Chang Gung University demonstrated gene recombination VP1 in Escherichia coli and used the purified VP1 protein as a subunit vaccine for immunoassay. (5) Professor He Meixiang also transferred the VP1 gene into the plastid to make a DNA vaccine, and then injected the DNA vaccine into the animal to express the W1 protein. 201100545 Hope that the VP1 can be expressed. Smoothly induce an immune response. (6) The attenuated strain of Salmonella typhimurium carrying the plastid representing the W1 protein, and the VP1 antigen expressed in the intestine of the mouse to induce a strong immune response as an oral live vaccine. (7) Using a genetically-transferred mouse, the state protein is expressed in the milk of the mother mouse as an oral antigen of the mouse. (8) Fanyou, who uses gene transfer, sees W1 protein as an oral vaccine in Fansu. (9) A chemically synthesized VP1 peptide (VP1 peptide) was directly used as an immunizing antigen to produce neutralizing serum. However, 'similar to (1) · (3) these attenuated viruses or deactivated viruses as a vaccine, the vaccine itself contains a complete viral gene, shame and disease safety considerations and deactivate the virus The vaccine also loses part of the immune-inducing ability during the treatment process. Like (4) (9), these directly use the purified ypj protein (or multiple wins) or use the vector to express the VP1 protein; the vaccine, the immune effect is not ideal. The overall effect is still worse than the deactivation of the virus vaccine. This may be due to the fact that Cong and VP3 also have neutralizing antigen mosquitoes, which are related to fine mosquitoes and impurities, and even some are located between different protein amino acids. W1 alone as a subunit vaccine may lose some importance. The epitope. Therefore, if you can use both, Cong, and vp4 as antigens and retain their original structure and shape in the wild virus, it is theoretically possible to induce more and stronger antibodies. The virus-like partide (VLP) is a shell-like structure composed of viral coat proteins. The 201100545 structure does not contain viral genomes, so there is no doubt about infection and replication, and it is safe to use. Because _ poison earns her with the real virus, it can cause the immune system in the body to produce specific antibodies to resist the invasion of real viruses and achieve the effect of protection. The traditional vaccines currently used include deactivated vaccines and attenuated vaccines, because the real virus is an external force to make the epitope unactivated or reduce its toxicity. The genetic material of the virus still exists in the vaccine, so it cannot be completely The opportunity to be infected after the vaccination is eliminated; compared with the former, the virus-like granules and the shells with similar real viruses can maintain antigenic sites close to the real virus, triggering a good immune response against the infection of real viruses. Therefore, the virus-like particle vaccine is a highly promising form of vaccine. Viral particle vaccines have been studied abroad, and most of the current virus-like particles are manufactured by baculovirus/insect cell expression systems, such as poliovirus, HIV, human parvovirus, and Chinese cystic virus. and many more. Some virus-like particles of clinically important viruses have also been shown, such as SARS coronavirus, hepatitis D virus, human papilloma virus, avian influenza virus H5N1 and influenza virus, etc., for more viral vaccine research. . The virus-like particles of human papillomavirus (HPV) are used as cervical cancer vaccines and have been produced by two pharmaceutical companies. Among them, Merck's Gardasil® is produced by the yeast system cerevisiae, which has been approved for marketing in Taiwan and the United States. Another cervical cancer vaccine product, CervarixTM, is a HPV VLP that was diagnosed by GlaxoSmithKline using the baculovirus/insect cell expression system.

More than 80 countries have been approved for listing. In addition, research on the use of viroid-like particles as a coating carrier to transport DNA or drugs into cells has begun to develop. It is pointed out in the literature that the efficiency of feeding genes into cells by virus-like particles is much higher than that of using microlipid or naked DNA 201100545. Therefore, in gene therapy, the gene therapy vector for sputum f particles is quite potential. In the case of enterovirus up to hundreds of serotypes, small silk fibrovirus has caused money for many years, and has been deactivated vaccine (Shaq) and attenuated vaccine (sand f) to prevent, but many other enteroviruses will not cause serious Clinical recording, so carefully study the financial "« unprotected seedlings. In particular, the EV system and the listed virus have been bribe late. So far, the other herpesviruses are still insufficient. However, the enterovirus 71 has been experiencing major outbreaks in China and other countries in recent years, so its virology, pathology and biology. The basic research has been greatly increased, and the information on gene composition and variability, pathogenicity, and immune virus information has been further understood. Therefore, it is quite helpful to develop new-generation vaccines and check-ups. However, as of now, the enterovirus 71 type of money has been the main slave to rely on the public health supervision system, including disease notification and P reading. Therefore, if it depends on prevention or treatment, the development of vaccines or antiviral drugs is extremely Important position. SUMMARY OF THE INVENTION Many literatures have reported many factors affecting the production of recombinant protein in insect mites/micro-paths, including the genetic design of baculovirus and the sensitivity of insect cell strains to baculovirus. The difference in the amount of downstream protein expression, the appropriate reactor parameters and the medium can effectively increase the yield of recombinant protein. A brief discussion of the effects between various basic parameters and the production of viroid-like particles. Firstly, the genetic design of the improved baculovirus of the present invention 'hope that the newly constructed baculovirus (Bac-Pl-C3CD) can make the insect cells have more resources to express the strong promoter by reducing the expression of the 3CD hydrolase'. 201100545 P1 protein downstream of polyhedrin. However, there are different distributions of extracellular virions in different cell lines. With Sf-9 cells as host cells, the strategy of using a weak promoter to drive 3CD to increase VLP production has its effect, but when it is produced with Ηΐ_5, it cannot increase yield. This is due to the strong sensitivity of Hi-5 cells to strong promoters, which makes the 3CD expression driven by the weak promoter too low. Although it can effectively express P1 protein in large quantities, it can complete P1 because there is not enough 3CD. Cut into W1, γρο, VP3, so that the production of viroid-like particles could not be improved. In addition, a small amount of trial production was used to discuss the cell concentration at the time of infection and the possible effect of M〇I on VLP production, and it was found that increasing the cell concentration did increase the VLP production significantly. Combining all the experimental results, it has been possible to infect Sf_9 cells at a high cell concentration (4 ><1〇6 ceiis/ml) using Bac-Pl-C3CD and obtain high-yield VLPs in the culture solution. The maximum yield of extracellular harvest is more than 5〇 mg/1, which is not only 5 times that of the past ## (about 10 mg/1), but also avoids the difficulty of the subsequent purification. The present invention provides a baculovirus translocation vector for expressing a viral protein, which comprises: (1) a strong baculovirus promoter and a - segment linked to the (tetra) acid sequence of the promoter and its side, wherein the nucleotide acid The sequence can be translated into a viral viral capsid protein; and (7) a weak promoter and a segment are linked to a nucleotide sequence downstream of the promoter and interacting therewith, wherein the acid sequence can be translated into a scaled virus shell egg (four) Eggs, wherein the strong baculovirus promoter drives a nuclear listener sequence linked downstream of the promoter to produce a higher protein yield than the weak promoter drives the protein translation translated by the (tetra) acid sequence downstream of the promoter. 11 201100545 The virus described in the present invention refers to enterovirus, and the most preferred embodiment is enterovirus 7i l. The strong baculovirus promoter described in the present invention is selected from the baculovirus polyhedrin promoter. The promoter or the P10 promoter, and the weak promoter is selected from the baculovirus immediate early starter, the cytomegalovirus promoter, or other vulnerable promoters that act in a baculopathic disease. The present invention also provides a recombinant baculovirus comprising a baculovirus translocation vector capable of expressing a viral protein which is used to infect a host cell to produce a recombinant protein, and a recombinant egg from a virus of f-intestinal virus Shell egg and hydrolyzed protein. The present invention further provides for the expression of a genus (4) transgenic silk host bacterium which is infected with a recombinant baculovirus. The baculovirus host cell described in the present invention is an insect cell, which is selected from the group consisting of Sf_9, a cell line, and the best embodiment is a Sf_9 cell line, and in addition, the host cell can enter the virus egg. The surface virus _ virus earns scales to the outside, while the viral protein is the viral shell protein and hydrolyzed protein of the enterovirus. The present invention provides a further step-by-step method for expressing silky filaments, which can express Pi, the recombinant domain, and the silk cell host cells to form a disease, and after the ringing, the supernatant is taken from 12 201100545 to obtain virus-like particles. The virus particles are viroid-like particles of enterovirus. The invention further provides a vaccine for preventing viral infection, which is selected from the group consisting of an immunologically effective amount of recombinant baculovirus, a host cell and a virus-like particle released by the host cell, and a pharmaceutical An acceptable immune adjuvant. The virus refers to enterovirus. [Embodiment] Materials Ο Cell culture insect cell culture The two insect cells used in the present invention are Sf-9 (Invitrogen, Cat. 11α 11496-015) and Hi-5 (Invitrogen, Cat· Νο_ Β 855-02), respectively. 〇Sf-9 cell line was treated with Sf-900 II (GIBCO, Cat No. 10902-088) and with 10% fetal bovine serum (Fetal bovine serum ' FBS, GIBCO) and 0_m (w/v) Pluronic F_68 ( Sigma)

TNM-FH (Sigma ' St. Louis ' MO) medium was cultured and suspended in an inner Q flask at a rotational speed of 90 rpm in a 5% c〇2 27 °C incubator. The cell line was Sf- The medium was cultured in 900 II serum-free medium and placed in 5% c〇2 of 2; the rc/m culture phase was cultured in suspension at spin at 90 rpm. In the present invention, the sf_9 cultured in the medium is used to amplify the m-virus of the baculovirus, and the Sf-9 cultured in the sf9〇〇II is subjected to the production of the enterovirus 71 type virus particles. Mammalian Cell Culture The mammalian cells used in the present invention are mainly human embryonic rhabdomyosarcoma cells 13 201100545 (Human Embryonal rhabdomyosarcoma, RD), and RD cells are contained in ι. /. The fetal calf serum and the PSN antibiotic were cultured in an Alpha minimal Essential medium (α-ΜΕΜ, Sigrim) medium. The cells were placed in a 5% C02 37 ° C incubator and incubated in a culture dish. The present invention uses RD cells mainly as host cells of enterovirus for the growth and thinning of enterovirus. Recombinant baculovirus recombinant bacillus disease The present invention uses the Bac-To-Bac® (Invitrogen, Carlsbad, CA) baculovirus expression system for virus preparation, and uses the pFastBac DUAL plastid in the system as the gene skeleton. Construction of a recombinant baculovirus. This plastid has two late-stage strong promoters of baculovirus, namely the polyhedrin prom〇ter and the p 1 〇 promoter, and each has a multiple selection downstream of the two promoters. The colony (Muitipie ci〇ning sjte, mcs I and MCSII) can colonize foreign genes into foreign genes. Therefore, according to the sequence of the published enterovirus strain TW/2272/98, the present invention connects the ρι gene carrying the viral coat protein to the downstream of the polyhedrin promoter, and the 3CD gene is ligated downstream of the pl〇 promoter, and the virus is named. For Bac-Pl-3CD. In addition, the present invention also links the 3CD gene to a weak promoter.

The cleavage knife was downstream of the baculovirus immediate early promoter and the CMV promoter (cytomegalovirus) and was named Bac_P1_I3CD and Bac_P1_C3CD (Fig. 1). Heavy-duty & disease-like females The fine-magnification of the re-transformation disease in the present invention is the use of Sf-9 cell line and 14 201100545 ΤΝΜ-FH medium with 10% fetal calf serum. When amplifying the virus, the sf_9 cell strain should be cultured in a spinner flask (Spinner Flask). When the cell concentration reaches about lxl〇6 cell/ml, the cells are centrifuged at 5 〇〇xg for 10 minutes, and 9 is removed. /10 of the medium was allowed to reconstitute the cells in 1/1 〇 original volume of medium. At this time, add the viral infection dose (multiplicity 〇finfecti〇n, Μ〇Ι) to 0.05 pfU / cell (defect plaque forming unit / cell, piaqUe f〇nr ^ ng unks per ceu) virus solution, uniform at room temperature After shaking for one hour, the medium was replenished with fresh medium and placed in a spinner in a 2rc incubator. After 4 to 6 days of infection, the survival rate of the fine cells was observed. When the cell survival rate was reduced to 60 to 70%, the cells were centrifuged at 3 Torr for 10 minutes to remove the cells and fragments thereof, and the supernatant was collected (ie, The virus solution is stored in the refrigerator in the dark. If it needs to be stored for a long time, it should be placed in a -80 °C refrigerator. End-point dilution method for quantification of viral effect 僧 End-point dilution method First, the disease is serially diluted 1G times with the nutrient, and then the Sf 9 cells with a concentration of 1><1〇5 eell/ml are 15() μ ΐ 〇 〇 〇 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 The collision 96 was reduced in human thorn culture incubator for I3 days. Under the optical microscope, the fine surface sensation was observed __, and finally the statistical method was used to calculate the virus titer. The virus concentration was called infection titer. (loud than t-shirt), the unit is pfo/ml (lytic plaque forming unit / ml, coffee ah f〇nning shirt π milliliter) ° Yue also calendar virus type 71 disease volume sea # 15 201100545 Enterovirus 71 type disease The sputum of the present invention uses the Sf-9 and Hi-5 cell lines to produce the EV71 VLP. When the cell concentration in the suspension culture reached about l-6xl06 cell/ml, the multiplicity of infecti〇n (MOI) was added to the virus solution of 0.001-, and the spiker was placed in a 27 °c incubator. In order to determine the optimal harvest time for the EV71 VLP, the cell fluid was sampled every 24 hours after infection and the VLP yield was analyzed by Western blotting. Bioreactor Production of Enterovirus Type 71 Viruses The present invention aims to increase the unit volume yield of viroid-like particles, and will attempt to produce viroid-like particles in laboratories and bioreactors after confirming optimal production conditions. The invention adopts

The Sartorius BIOSTAT® B fermentation tank is produced. BIOSTAT® B is an agitated bioreactor that is vented using a bubble-free ventilation system. The invention adopts an electronic gas mixing device to regulate the mixing ratio of nitrogen, oxygen and air, and increases the dissolved oxygen amount by blowing air in the air portion of the tank body, and regulates the rotating blade speed at 1〇〇φΠ1 to control the temperature. At 27 degrees. The enemies of the intracytoplasmic EV71 virus will be centrifuged for 72 minutes after infection for 72 minutes (R123Arotor, 11000 rpm, 18000 xg 'CR22, Hitachi) to collect the cells. After removing the medium, the cells are returned. Dissolve into TNE buffer (40 mM Tris, 1 〇〇 should be NaQ, 2 mM MgCl2, 丨 mM EDTA, 卩 117.4) in 'Set-80 (1! Ice phase, use repeated ice; East / solution; East (? 1恍2£311(^111&\¥) three times to break the cells. Then add l/io volume of tissue decomposition solution (lysis buffer, 1% NP-40, 40 mM Tris '100 mM NaCl, 2 mM MgCl2) ' 1 mM EDTA, pH 7'4), 16 201100545 The reaction was allowed to stand on ice for 30 minutes. The cell solution after the reaction was centrifuged for 30 minutes (RA202 rotor '11000 rpm '18000 xg, CR22 'Hitachi), and the supernatant was collected. At this time, the supernatant solution contains the virus-like particle protein. The extracorporeal enterovirus type 71 disease rounds are collected and the Sf-9 cells at different time points (such as 72 or % hours) after centrifugation are centrifuged for 30 minutes. (R123A rotoi* 'll, 〇〇〇ipm, 18,000 xg 'CR22 ' Hitachi) After receiving the supernatant.鎞 类 类 鎞 澧 澧 澧 澧 澧 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置〇〇μ1 was mixed with 1〇〇μ1 protein stain (BioRad protein assay; BioRad, CA) and placed at room temperature for 10 minutes. The absorbance at 595 nm (〇D) was measured to prepare a standard. BSA calibration curve (25 pg/mM.5625 pg/ml). At the same time, the virus-like particle sample obtained by ultra-high-speed centrifugation of the present invention is diluted by h2〇, and then taken 4〇〇μ1 and 1〇. The protein dye (BioRad) is uniformly mixed, placed at room temperature for 10 minutes, and its absorption value at 〇ε > 595 is measured. The total number of virus-like particles can be estimated from the calibration curve drawn by the absorbance of the BSA standard. Protein concentration. Purification of enteric disease volume 71 This invention uses enterovirus type 71 as TW/2272/98 ( belonging to gen〇type 〇: 2) and gen〇type Bh TW/2272/98 as the first outbreak of enterovirus in Taiwan in 1998. The virus strain isolated during the pandemic, and genotype B5 is the main cause of enterovirus epidemics from 2007 to 2008 17201100545 no strain 'Cloth after gene sequence confirmed genotype Bs. In the experiment, milk cells were used to carry out the cultivation of all virus strains. The virus was as follows: when the ribs were aged 15 coffee cultures to about 80% full, the original culture solution was removed and 15 hours of Phosphate-buffered saline was added. The (PBS) buffer was used to wash the residual culture solution, and after washing the cells, the PBS was removed. Further, in the present invention, 1 〇 ml of the α ΜΕΜ culture medium containing no fetal bovine serum is diluted with the enterovirus 71 sterilizing solution, and the diseased surface is cultured, and then the culture disk is placed on the side of the shaking plate for 1 hour for virus infection. After adsorption by the sputum virus, the α-ΜΕΜ culture solution containing the diseased sputum was removed, followed by the α-ΜΕΜ culture medium containing 2% fetal calf serum and pSN antibiotic, and cultured in a 37 ° C incubator. When 7〇% of the cells produced a typical cytopathy effect (CpE) such as cell enlargement and shedding, the infected culture solution was collected and centrifuged for 10 minutes (5000 rpm), and the supernatant was collected after centrifugation. In addition, the infected cells and culture medium were collected, and Can_4〇 (final concentration 1%) was added to the solution to help release the virus from the cell fragments, and the cells were removed by two freeze/daw methods. break in. After the cell fragments were removed by centrifugation at 6000 rpm for 10 minutes (4 Torr x R20A2 rotor), the virus-containing solution was stored at -8 Torr. (: Or directly purify. When purifying, place the sample in a 4: (with an environment of stirring), slowly add sodium vaporate to a concentration of 2% (w/v) in a continuous stirring environment, and gasify. After the sodium is completely melted, add polyvinyl butanol 8000 (PEG 8000) to a concentration of 14% (w/v). (Alternative method: 60% (w/v) PEG 8000 solution in XNE buffer as solvent, slow Add the virus solution to the concentration of PEG 8000 (14% (W / V)). After the PEG 8000 is dissolved, leave the sample in an environment of 4 ° C and continue to stir overnight, using PEG 8000 will precipitate the virus. After completion, the 18 201100545 supernatant and the sediment were separated by centrifugation at 6000 rpm for 30 minutes (R20A2 rotor), and the sediment was reconstituted with 1/10 of the original virus volume of the top buffer. The ruthenium was dissolved in TNE buffer. , respectively, configured as a solution with a concentration of Μ%, 4〇% (Wv) by weight, and then with a syringe and a 18-gauge long needle (in the order of 25%, in the order of qing), the solution is injected into the volume from the bottom. In the ultra-high speed centrifuge tube of n ml, the volume is 5, 3 ml respectively; when the high concentration solution is injected from the bottom, it can It is enough to raise the low concentration of the vegetable sugar solution, which can reduce the mixing of the solution between different concentrations. After completing the 25. sugar gradient, the sample of Na 5 ml is carefully added to the uppermost layer of the solution in the tube. After completing the above (4), you can hang up the Swinging bueket and centrifuge at 1000°g for 4 hours (P40ST rotor, 30,000 melon, 113〇〇〇xg,

CP100MX ’ Hitachi). Then slowly suck the solution from the bottom with a syringe and a long needle, collect a tube every 0.5 ml, and analyze the content of πρ in each tube by Western blotting. The virus will be located at the interface between 25% and 40%. The needle collects the band of the interface. After the analysis, the fraction containing the VLP was concentrated and diluted in about 10 times the sample volume of TE buffer' and then ultracentrifuged at 3 rpm for 2 hours (i〇〇〇〇〇xg P4〇ST 〇rotor) The enterovirus is precipitated by centrifugation; the supernatant is removed, and the precipitate of the bottom portion is added by adding appropriate PBs buffer, and the insoluble portion is removed by centrifugation at 450 〇 x g for 10 minutes (Universal 32). That is, it contains the purified enterovirus. The purified virus was analyzed for virus titer according to the endpoint dilution method. Viral titers are determined by end-point dilution based on the principle of typical cellular pathological changes (CPE) of infected cells. The resulting virus titer unit is TCID50. The experimental method is to culture the RD cells in a 96-well cell culture dish. When the cells multiply to about 80% full, the virus to be tested is serially diluted by 10 times with α-ΜΕΜ culture medium containing 2% fetal bovine serum and PSN antibiotics ( l〇_2-l〇_1G), extract the original 201100545 culture solution, take (10) μΐ _ release county _ fox, infect at least 12 holes per dilution rate. After infection, the cells were cultured for 3 days in a 37 °c, 5% c〇2 incubator. After the infection, the fine CPE _ paste final (10) interpretation formula was observed. Example 1 yi^p characterization analysis Westem blotting Transfer of the separated SDS-PAGE white matter sample to the wall using a vertical transfer tank (Mini Trans-Blot Cell; BioRad, Munich, Germany) Fiber membrane (〇45μηι

Mitroeellulose membmne, MC membmne; Pall Corporation). Before the transfer, the NC membmne 'and the miscellaneous-cutting buffer (2.5 mM Tris, 192 mM glycine, 20% v/v methanob pH 8.3) were transferred to the transfer buffer (2.5 mM Tris, 192 mM glycine, 20% v/v methanob pH 8.3) for five minutes. The NC membrane was placed on top of the gel and a filter paper infiltrated with transfer buffer was placed on top of each other with a transfer current of 1 〇〇v for 6 〇 minutes. The transferred membrane was placed in a blocking buffer (25 mM Tris, 150 mM NaCl '5 ° / 〇 nonfat powdered milk 'pH 7.4) at room temperature for one hour or 4 〇 c overnight. Next, a 1500-fold dilution of mouse anti-VP1 monoclonal antibody was used as the first-order antibody, and the VP1 protein on the membrane was identified and allowed to act at room temperature for 2 hours or 4. (: function overnight, then wash three times with washing buffer (TBST ' 0.05 ° / 〇 Tween-20 in TBS) for about 10 minutes each time to dilute 2500 times Alkaline Phosphatase-conjugated goat anti-mouse

IgG monoclonal antibody (Chemicon, Temecula, CA) as a second-order antibody, recognizes the capture of the first-order antibody, and acts at room temperature for one hour' followed by the coloring agent 5_bromo chloroindoxyl 20 201100545 phosphate (BCIP) / nitro blue tetrazolium ( NBT) (Sigma) is colored. Enzyme-Linked Immunosorbent Assay ELISA can be divided into two methods: direct and sandwich. The present invention adopts the sandwich ELISA method to improve the accuracy. The antibody for adsorption (mystery (10)-milk-multiple antibody) was diluted 1:10000 to each well of 100 μl to ELISA 96-well microtiter plate (Nunc ELISA/EIA Maxisorp; Nunc, Roskilde, Denmark) The antibody was adsorbed to the bottom of the well by placing it at room temperature for 2 hours or at 4 °C overnight. After the adsorption was completed, the solution was aspirated and poured into each well with 100 μΐ of the washing solution PBS-T (0.05% Tween 20 in PBS buffer). After standing at room temperature for 10 minutes, the washing solution was taken out, and the washing step was repeated 4 times. After the last removal of the cleaning solution, add 1 μl of blocking buffer (PBST solution containing 1% BSA) to each well for 1 hour at room temperature, fill all the voids in the bottom of the well with protein, and then wash repeatedly with washing solution. 4 times. After removing the cleaning solution, add ι〇〇μι after dilution (sample dilution ratio 1:1 〇〇) sample to be tested (such as cell fluid containing virus-like particles) 〇 or enterovirus type 71 virus particle standard (standard The product is diluted from 0.5 pg/ml in two-fold sequence until 0.015625 pg/ml). The standard is obtained by ultra-high-speed centrifugation of viral particles such as intracellular production, and reacted at room temperature for 2 hours, followed by a cleaning solution. Repeat the washing 4 times. After removing the washing solution, '100 μl diluted EV71 monoclonal antibody (mouse igG2b monoclonal antibody, Cat No. 3324; Chemicon, Temecula, CA) was added, and reacted at room temperature for 2 hours, followed by repeated washing with washing solution 4 Times. After removing the washing solution, add 100 μM, diluted 2000-fold g〇at anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories 'Gaithersburg, MD) secondary antibody, and react at room temperature 21 201100545 for 1.5 hours. The cleaning solution was washed repeatedly 4 times. At the last wash, TMB (3,3',5,5'-tetramethyl benzidine; Sigma) was prepared and 1 mg of TMB was dissolved in 1 ml of dimethyl sulfoxide (DMSO; Sigma), followed by 9 ml. Phosphate-Citrate Buffer (0.1 M NaH2P〇4 and 0.1 M citric acid, adjusted to pH 5 with bowl acid). After removing the washing solution, 100 μM TMB (Sigma, cat# T0440) was added to each well for color reaction. The reaction was carried out at 37 ° C for about 15 minutes until the reaction reagent showed a distinct blue color. Then, 5 μM 2 M H2S〇4 was added to each well to stop the color reaction, and the micro 1 titration plate was placed in an ELISA reader (MRX; Dynex Technology Inc., Glen d Allen, VA) at a wavelength. The ELISA values were read at 450 nm. According to the concentration of the viroid particle standard and the obtained absorbance value, after the calibration curve is prepared by the present invention, the virus-like particles in the sample to be tested are relatively quantified. Transmission Electron Microscopy (TEM) In the present invention, the purified VLP sample is submerged under the centrifuge tube by ultracentrifugation, and then dissolved back by loo μΐ ddK2. VLP-like mouthwash after purification of 1〇μ1 (concentration adjusted to about 50pg/ml) at TEM copper mesh (support films formvar / carbon 200 mesh copper grids,TEDPellaInc,cat# 〇18〇〇 Above _F), after standing for 5 minutes, apply filter paper and dry excess droplets. Take 1〇μ1 of 2% ph〇Sph〇tungStic acid (ρτΑ, Sigma, adjusted to pH 7, 2 with NaOH) and drop it on the copper grid for 3~5 minutes. Finally, filter the excess dyeing agent with filter paper. Place the copper _ face up and place it on the cold air towel under dehumidification. After air drying overnight, you can directly observe the electrons (10) aehi, H_75 (8). 22 201100545 Immunogold staining and transmission electron microscopy observation In the calibration experiment of immunogold particles, the present invention first placed jjgM copper mesh on parafilm, about 10μιη (concentration of 5〇μβ/ηι1) The virus-like particle sample was dropped on a carbon-coated copper mesh, and after standing for about 5 minutes, the excess sample was sucked off with a cotton paper, and then the phosphate buffer saline (PBS) was dropped on the carbon-coated carbon. The excess protein was washed off the copper mesh and excess PBS was blotted with tissue paper, and the washing step was repeated three times. After washing, the anti-VP1 antibody was diluted with PBS (1:2 〇〇 dilution), and the _ diluted droplets were allowed to stand on the copper grid for 1 hour to allow the antibody to be adsorbed onto the sample. After the end of the antibody reaction time, the above washing method was repeated, and the surface of the copper mesh was washed three times with PBST, followed by the secondary antibody.

Cg〇ld_conjugated goat anti-mouse IgG, Sigma) diluted 1:1000 in PBS' Take diluted antibody 1〇μ1 drops on copper net, connect the size of the secondary antibody to $ crispy gold particles, age Under the electron microscope _ presentation. After the tertiary antibody was reacted for 1 hour, the surface of the copper mesh was washed three times with PBST. After washing, the surface solution of the copper mesh was blotted with cotton paper, and a solution of 1〇μ1 of 2〇/capric acid crane was dripped in Tongshe, and the dyeing was allowed to stand. After three minutes, the excess dyed hair was dried with cotton paper and placed for -12 hours, as observed by an electron microscope (H-7500). The combination of the strain and the cell strain was learned from previous laboratory studies that the rod-shaped silk/insect filaments together express the protein Pi of the enterovirus type 71 (driven by the p〇lyhedrin strong promoter) and 3CD (with pi Reluctant promoter drive) 'P1 can be 3CD _ 丨 纲 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The baculovirus prepared was named Bac-Pl-3CD (see Figure 1). To enhance the production of viroid-like particles, the present invention contemplates that a small amount of 3CD hydrolase can cause the P1 protein to be cleaved and form viroid-like particles. Therefore, the present invention replaces the P10 promoter with a weaker promoter (IE-1 and CMV promoter) in insect cells. It is desirable to increase the amount of P1 protein by reducing the amount of 3CD hydrolase, and further improve the class. The production of viral particles. Therefore, the present invention also ligates the 3CD gene to a weak promoter such as the immediate early promoter and the CMV promoter (cytomegalovirus) to prepare two other viruses, Bac-Pl-I3CD and Bac-Pl-. CBCD (see Figure 1). Recombinant baculoviruses Bac-Pl-3CD, Bac-Pl-C3CD, and Bac-Pl-I3CD can express P1 and 3CD recombinant proteins. After the construction of the baculovirus strain, 'Sf-9 and Hi-5 cells were cultured in Spinner Flask by suspension culture method', when the cell concentration reached about lxl〇6 cell/ml, Bac_P1_3CD, Bac-Pl-C3CD and Bac-, respectively. Pl-I3CD is infected with MOI10. At 48, 72, and 96 hours after infection, the cell fluid was centrifuged at 10 μg for 10 minutes, and the VP1 protein expression was initially detected by Western blotting. In the case of sf-9 cells, comparing the vpi production of three baculoviruses in Figure 2(a), it was found that regardless of the cell supernatant collected on the day after infection, the Vp protein production by Bac_p丨 was The yield was lower than that of the recombinant baculoviruses Bac_pi_C3CD and Bac-Pl-l3CD. Bac_pi_C3CE^Bac_pi i3CD achieved the highest W1 yield on the fourth day after infection, and Bac_pi_C3CD produced the highest extracellular νρι yield. Then, (4) on the production of viroid-like particles, the insects with higher performance were analyzed, and the virus infection and cell supernatant were analyzed by the experimental conditions of Sf-9 _ 24 201100545. The results of the experiment were analyzed by Western blot as shown in Fig. 2(b). Conversely,

After infection of Hi_5 cells, Bac-Pl-C3CD and Bac-Pl-I3CD produced much less VP1 protein than Bac-Pl-3CD, and the expression of VP1 protein gradually decreased with time. However, the expression of VP1 protein infecting Ηι·5 with Bac-Pl-3CD gradually increased with time. In order to know which baculovirus and insect cell strains are used, the highest virus-like particle yield can be obtained. The present invention further improves the number of experimental groups with better expression in Western blot results.

❹Analysis was performed by ELISA. Three strains of virus were infected with Sf_9 cells, or Hi-5 cells were infected with Bac_pi 3cD for a total of four experimental conditions. The supernatant was analyzed by EUSA on the fourth day of infection.

The amount of expression of the inner type of virus particles. The results are shown in Figure 2(c). The yields of extracellular viroid particles infected with Bac_pi_3CD and Bac-Pl-I3CD were 1.5 mg/L and 7.8 mg/L, respectively, while the BaC-P1_C3CD group had the highest yield. Up to 12 5 mg / L. If yang is the host cell, the Bac-Pl-3CD group can reach 12.1 mg/L. The VLP production by the two groups of infected cells and virus combinations was better and the results were similar. Therefore, the present invention selects two cells and virus 、, combination (infecting Sf-9 cells with Bac-P1_C3CD and infecting Hi-5 cells with Bac-Pl-3CD) for optimal concentration of infected cells (CCI, Cell Concentration at Infection) Conditional discussion. In addition to improving the genetic design of baculovirus, the effect of cell concentration at the time of infection on Mp production was also investigated. First, the present invention infects cells infected with Bac_pi_C3CD at a concentration of 1χ106, 2χ1〇6, 4χ1〇6, and 6xl〇6 Cells/ml, and the cell supernatant on the 4th day after infection is taken. Liquid, extracellular viroid particle yield was analyzed by ELISA. As shown in Figure 3 25 201100545, when the cell concentration increases, the production of viroid-like particles also increases, and the highest yield can be 43. 3 mg/L when produced at 4χ1〇6 cells/ml. However, excessive cell infiltration (6χ1〇6 cell/ml) may result in a rapid decrease in cell viability due to a shortage of nutrients, which in turn may result in a decrease in the production of virulence particles. In the Hi-5 part, since the maximum cell concentration of the Hi-5 cells cultured in SF-900II in the spinner flask is 3x10 cells/ml, the infection is carried out at a cell concentration of 1χ1〇6 and 2xl〇6cell/m丨, and The cell supernatant was collected on the 4th day after infection, and the extracellular ◎ viroid particle production was quantitatively analyzed by ELISA. As shown in Figure 3, increasing the concentration of cells at the time of infection at a fixed dose (M〇I 1〇) did increase VLP production, similar to the results of Sf-9 cells. When the cell concentration reaches 2xl06Cell/ml, the highest amount of virus-like particles can be 22 2mg/L. Taken together, the Sf-9 cells were infected with M〇I 1〇 Bac-Pl-C3CD virus at a cell concentration of 4×10 6 cdl/ml, and the maximum yield was obtained on the fourth day. Therefore, it was decided to infect under this condition, and the supernatant was collected on the fourth day as a production parameter for the production of virus-like particles in the subsequent reactor. The cell fluid collected by this infection condition was also centrifuged in a high-speed centrifuge for 30 minutes (18,000 rpm), and then the supernatant was collected and subjected to preliminary purification as an antigen for subsequent mouse experiments. Reactor production of virus-like particles After determining the optimal production conditions for viroid-like particles in a rotating angle bottle, an attempt was made to produce virus-like particles using a laboratory grade stirred bioreactor (BIOSTAT® B). Different dissolved oxygen values (DO 15%, 30%, 60%) were adjusted in the experiment to compare the effects of different dissolved oxygen levels on the production of extracellular disease 26 201100545 toxic particles. The same production conditions but the production of viral particles produced in a rotating angle bottle were used as a positive control. The results of the ELISA analysis (Fig. 4) showed that the production of viroid-like particles produced in the reactor was higher than that produced by the virulet-like particles produced by the rotary angle bottle. In addition, the amount of dissolved oxygen is controlled at 30% of the virus-like particles, and the maximum yield after the fourth day is 64.3 mg/L. Therefore, in the portion of the reactor dissolved oxygen, the dissolved oxygen content was selected to be 30% of the reactor production parameters. Qualitative analysis of viroid-like particles The production of viroid-like particles produced with Bac-Pl-C3CD was significantly increased compared to the previous production of viruvirus particles produced as Bac_pi_3CD. In order to confirm that the viroid-like particles produced by Bac-Pl-C3CD have the same basic properties as the viroid-like particles produced by Bac-Pl-3CD, the present invention infects Sf-9 cells with Bac-Pl-C3CD to produce viroid-like viruses. The granules were the experimental group, and the viroid-like particles produced by infecting Hi-5 cells with Bac-Pl-;3CD were positive control groups, and subsequent qualitative analysis was performed. The virus-like particles after centrifugation (including the experimental group and the positive control group) were negatively stained with phosphotungstic acid and observed by a biotransmission electron microscope. It can be seen from Fig. 5(a) that the VLP purified by the present invention has a circular shape with a particle size of about 25 Å, which is similar in size to the actual virus particles (27 nm) and is previously infected with Bac-pi_3CD. After twisting 5 cells, the VLP (Fig. 5(b))' obtained from the age field and from the PEG dirty knot _ lang method was too different in appearance and size. The black space between the rags may be caused by the people inside the granules, so __ Ying County's hollow granules. 27 201100545 To further demonstrate that these granules are enterovirus 71 VLPs, the present invention recognizes and captures VP1 protein on enterovirus VLPs with anti-VP1 monoclonal antibodies, and then recognizes secondary antibodies linked to gold particles (5 nm). The VP1 monoclonal antibody was taken, and the immunogold label thus completed was observed by a transmission electron microscope, and the results are shown in Fig. 5(c). The distribution of VLP can be vaguely seen in the figure, but it may be due to the use of more antibody proteins, and the appearance of the particles becomes more blurred compared to the simple negative staining results. It can be clearly seen that the small black dots of 5 nm gold particles are mostly surrounded by viroid-like particles, so it is confirmed that these particles contain the enterovirus VLP protein VP1. At the same time, the experimental results were compared with the previous laboratory infection of Sf-9 cells with Bac-Pl-3CD. After breaking the cells and purifying the virus-like particles by ultracentrifugation, the same antibodies were used for immunogold labeling and electron microscopic observation. The results were compared, as shown in Figure 5(d). Gold particles were also found to surround the viroid-like particles. Based on the above results, the particle size and shape of the two types of virus-like particles were observed by a transmission electron microscope, and it was confirmed from the immunogold staining that W1 was located on the surface of the viroid-like particle. It can be concluded here that the virulence particles produced by infecting Sf-9 cells with Bac-Pl-C3CD are very similar to the basic properties of virulent granules produced by laboratory cells in BaoPUCD _HU _ or qing cells. Example 2 Analysis of iA immunoassay Mouse immunization and serum sample collection In order to understand the immunological properties of purified viroid-like particles, the present invention conducts animal experiments by means of intraperitoneal injection. After the sample was purified, the total protein concentration was adjusted to 40 pg/ml and stored in a -80 °C refrigerator. Female BALB/c mice are ordered by the National Animal Center and sent to the Animal Hall of Qingda for quarantine. The mice are injected intraperitoneally at 6-8 weeks of age. Before the intraperitoneal injection, the sample was mixed with the same volume of adjuvant (complete Freund's adjuvant for priming injection, Sigma >cat#F5881; incomplete Freund's adjuvant for booster injection, Sigma, cat# F5506) in a three-way valve for about 3 minutes. The sample was uniformly mixed in a white latex-like e. Each mouse was intraperitoneally injected with a latex sample of 500 μΐ (1〇total protein/mouse), which was indicated as the third week. At the fourth week, booster injection was performed in the same sample preparation method and injection method (but the adjuvant was incomplete Freund's adjuvant). At the 7th week, the blood of the mice was collected by facial blood collection, and the blood of the collected mice was placed in a 4 C refrigerator for 4 hours, and then centrifuged at 2 Torr x for 1 minute. The obtained clarified mouse serum was deactivated at 56 ° C for 30 minutes, and then subjected to subsequent analysis. The experimental group consisted of the following five groups: (1) PBS group: In order to eliminate the effect of the adjuvant on the experimental values, the present invention carried out the PBS-aged ship, and used the collected serum as the basis for numerical evaluation. (2) Negative control group (rBV): In order to understand whether the antibody reaction is indeed caused by the virus-like particles, rather than the errors caused by other miscellaneous proteins, the present invention does not carry any exogenous egg from the genus of the rice worm virus Sf_9 The secret was purified by the same purification procedure as the experimental group, and the protein at the same position was collected as a negative control group. (3) Positive control group (EV71) - Enteric virus obtained by purification by sucrose discontinuous gradient method The purified enterovirus still has an infectivity, so it is activated by heating the muscle for a minute. (4) Positive control group (BaC-P1-3CD): The virus particles were produced by infecting mouse cells with Bac pore 3CD. (5) Experimental group (Bac-Pl-C3CD): Sf-9 cells were infected with Bac-Pl-C3CD to produce viroid-like particles. The number of experimental animals per group was only four. Antibody titer First, configure the Coating buffer; first prepare 0.1 M Na2HP04 (Riedel-de Haen ’ cat# 30414) (pH about 9.0) and 0.1 MNaH2P04 (Lian Gong, cat# 90093) (pH about 4.5) with ddH20. A fixed amount of 0.1 MNa2HP04 solution was adjusted to pH 6.8 with 0.1 MNaH2P04 to obtain a Coating buffer (pH 6.8). Next, the purified enterovirus was quantified by protein assay, and diluted to a total protein concentration of 5 μθμΐ with a Coating buffer. Then, 1 μμΐ was added to a 96-well plate, and the mixture was incubated at 37 ° C. One hour was allowed to adsorb the antigen to the bottom of the well. After the adsorption was completed, the solution was aspirated, and 200 μL of the washing solution PBS-T (0.05% Tween 20 in PBS buffer) was injected into each well, and after standing at room temperature for 3 minutes, the 'cleaning liquid was taken out'. The repeated washing steps were repeated 3 times. . After the last removal of the cleaning solution, 100 μΐ of blocking buffer (0.1% BSA in PBST) was added and the reaction was allowed to stand at 37 ° C for 1 hour. 'All the voids in the bottom of the well were filled with protein, and then washed repeatedly with a cleaning solution. 3 times. After the washing solution was taken out, the serum of the mouse diluted with 1 μμ1 was added (two-fold serial dilution in eppendorf with a blocking buffer, dilution ratio of 25 to 22Q), and the reaction was allowed to stand at 37 ° C for 1 hour. After repeated washings with washing solution for 3 times, a goat anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories > Gaithersburg 'MD) secondary antibody diluted 5,000 times was added, and the reaction was allowed to stand at 37 for 1 hour. Then repeat the washing 3 times with the cleaning solution. Then, 100 μΐ/well TMB (Sigma, cat# T〇440) was added for 30 201100545 coloring, and the reaction was carried out at room temperature. After the appearance of a clear blue color, 5〇_2M ΗΑ〇4 was added to each well to stop the color reaction, and the microtiter plate was placed in EusA (Brain (3) Technology lnc., Glen Allen, VA). Read the falsified value with the wave mo leg.

After reading the absorbance value (OD), first check the 〇D value of the negative group (injected pBS group) 疋 No i at 0.2 ’ If it is greater than 〇.2, it should be judged that the test is invalid and need to be retested. The positive group was judged to be a 〇D value greater than the negative group. For example, if the absorbance 阴性 value of the negative group is 〇.2 ’, then if the experimental group is diluted to 216 and the absorbance is lower than Ο.4 (0.2+0.2), the titer of this experimental group is judged to be 15. Neutralization titer First, the attached RD cells were desorbed with Trypsin, diluted to 2χ1〇5 cells/ml′ and the suspended cells were seeded into a 96-well plate, placed at 37°c of 5% c〇2. In a constant temperature incubator overnight. The deactivated mouse serum samples were then serially diluted 2 times (23~2 ls) with 2% FBS and 1% brain full valence α-ΜΕΜ, and 〇 with the same volume of 2000 TCID50live EV71 (mixed After 1000 TCID50 EV71), the mixture was mixed and placed in a 37 ° C incubator for neutralization for 1 hour. Next, the overnight cultured milk cells were taken out from the 37 <3C incubator, and the culture solution was removed, and then the serum virus mixture which had been reacted for 1 hour with ΙΟΟμΙ/well was added, and then placed in a 37 °C incubator. After 3 days, the condition of cytopathic effect (CPE) in the 96-well plate was observed. According to the case of CPE, it is possible to protect 5 〇 % weu from the serum dilution ratio of CpE, which is the neutralizing antibody valence of the serum sample. For example, the subsequent dilution ratio 31 201100545 group neutralization reaction is stopped (the number of CPE wells is greater than 50 ° / 〇) ' then 211 is the neutralizing antibody calibration of the serum sample. Cross-Reaction Analysis The present invention utilizes an antibody neutralization assay to analyze the effect of mouse sera induced by KV3 strain VLP on neutralizing the infectivity of different strains of enterovirus 71. The experimental method was similar to the neutralization test. First, the attached RD cells were desorbed with Trypsin, diluted to a concentration of 2×10 5 cells/ml, and the suspension cells were seeded into a 96-well plate (ΙΟΟμΙ/well). The deactivated mouse serum was then serially diluted 2 times (23 to 215) with α-ΜΕΜ containing 2% FBS and 1% PSN antibiotic, and the diluted serum was mixed with enterovirus 71 of different serotypes ( For example, TW/2272/98 strain and Β5 strain) 'All virus doses used are 100 TCIDsq, and the mixed sample is reacted at room temperature for about 丨 hours. After the reaction is completed, the culture solution in the 96 well culture plate is removed, and then A mixture of 1 μl mouse serum and the virus to be tested was added to the culture well (final virus concentration was 100, and the cytopathic phenomenon in the 96-well plate was observed after culturing for 3 days in a 37 τ incubator, and according to the The neutralization titer determination method is used to determine the serum neutralization titer. The statistical analysis of the numerical difference analysis method is performed by using at least three repeated experiments, and the heart test (S-Mest) is used for statistical analysis. ("-) is less than 〇〇5 means that there is a difference between the values'. If it is greater than _, it means that there is no significant difference on the number side. Results 32 201100545 Lotus virus particles as a vaccine for 疬 y y antibody titer analysis in order to determine the VLP Immunology Whether the latter mouse can produce mfEVW-specific antibodies, firstly, the mouse serum collected by the experiment was heated at 56 ° C for 3 minutes to activate the interference to remove complement, followed by antibody titer analysis. The PBS negative control group did not elicit an antibody immune response, while rBV elicited a slight immune response with an antibody titer of approximately 27. However, the experimental group (Bac_P1_C3CD) and the two groups of positive control groups (EV7i and Bac_pi 3cD) had anti-antibody antibodies. The value can reach 212-213, and there is no statistical difference (4) 〇 5). Neutralization titer analysis of antibody titer can only analyze the ability of antibodies to recognize enterovirus antigens, and can not represent The ability of the antibody to neutralize the real enterovirus during actual infection, so the ability of the antibody to neutralize the real virus is evaluated by serum towel and titer analysis. The present invention serially dilutes the mouse serum by a 2-fold dilution (minimum dilution ratio is 23), and Mixed reaction with 1 〇〇TCI〇5Q real EV71 (TW/2272/98 strain) Ο Two hours later, RD cells were infected by adding 96-well plates, and CPE phenomenon was observed after three days, and the neutralizing titer was calculated. Figure 7(a) The results showed that the PBS and rBV groups in the negative control group and the lowest dilution tree could not respond to the EV71, and the neutralization titer of the EV71 in the seventh week after injection was 27, while the positive control group Bac-Pl. -3CD and the experimental group Bac-Pl-C3CD have a good ability to neutralize the real EV71, and the synergistic value can reach 21α75 and 21α5, respectively, and there is no statistically significant difference between the two (household > 〇 5). It is estimated that the neutralizing titer caused by deactivated enterovirus is lower than that of the experimental group Bac_pi_C3CD or the positive control group Bac-Pl-3CD may be the purity of the mechanized poisoning, resulting in no immune response caused by the 2011 201100545 method. Cross-protection of antibodies against different enterovirus strains The live virus (TW/2272/98) used in the aforementioned neutralization titer experiment is identical to the disease used in the production of virus-like particles of the present invention, and is all retroactive. Virus strain. And the enterovirus that was popular in 2008? ! The type belongs to. In order to confirm that the antibody induced by the drug is a shawl and (4) a gene-raising virus type 71, the mouse blood/month collected by the crane was neutralized for the strain of genotype B5. First, milk cells are used to infect proliferative diseases, and the concentration of each virus is measured. The neutralization test of the virus is used to determine the neutralizing intensity of the virus induced by the virus-like particles. From the results of Fig. 7 (6), similar results to Fig. 7(a) were found, the negative control group PBS and Lin group could not effectively neutralize enterovirus 71 type, while the positive control group EV71 induced species and titer of π5, still lower than positive. The control group BaC-Pl-3CD (potency up to 2 hearts), and the antibody induced by Bae_pi_C3 (:D-produced virus-like particles, the highest dilution ratio of the neutralized B5 strain can reach 2, which represents Antibodies induced by strains of enterovirus 71 virus can also effectively reduce the infectivity of other different enterovirus 71 strains. The available data show that 'virus particles obtained in a newly established way and this experiment The previous production method of the _ county has a good potential as a candidate for the W-type vaccine in the intestine. [Simplified illustration] The genes P1 and 3CD are separately transferred into the construction of the recombinant baculovirus vector of Fig. 1. EV71 34 201100545 The pFastBacTM DUAL plastid can be made into a virus (a) Bac-Pl-3CD (using the pi〇 promoter to drive 3CD) after the Bac-to-Bac System recommended experimental procedure, (b) Bac-Pl -C3CD (with CMV promoter driving 3CD performance) (c) Bac-Pl-I3CD (expressed by the IE-1 promoter to drive 3CD). Figure 2 Comparison of three strains (Bac-Pl-3CD, Bac-Pl-C3CD, Bac-Pl-I3CD) in different cells Production of Enterovirus 71 virus-like particles · When the cell concentration is about 〇6 〇cell/ml 'Infected cells with MOI10' 2 days, 3 or 4 days (days post-infection, dpi) The liquid was centrifuged at low speed to collect the supernatant as a sample of extracellular viroid particles. (a) Analysis of the expression of Sf-9 cells by Western blotting method. (b) Analysis of Hi- by Western blotting method 5 cell expression. (c) ELISA for quantification of different gene recombinant baculovirus in infected Sf-9 cells (□) ' and 72 hours after infection of Hi-5 cells (_) with Bac-Pl-3CD Total yield. The above experiments were all carried out in 1 〇〇ml spinner flask. The medium used in the experiment was SF-900II. The experimental results were three independent cells.

The average of the CI W culture experiments. Figure 3. Effect of different cell concentrations on the production of enterovirus 71 virus-like particles. When the concentration of Sf-9 cells (□) was about 1-, 2-, 4- or 6x106 cells/ml, cells were infected with Bac-Pl-C3CD (MOI 10). Alternatively, when the concentration of Hi-5 cells () reached 1 or 2 x 106 cells/ml, cells were infected with Bac-Pl-3CD (MOI 10). Samples were taken 72 hours after infection, the cells were centrifuged, and the supernatant was collected to quantify the concentration of viroid-like particles by ELISA. All of the above experiments were performed on a 100 ml spinner flask, and the medium used in the experiment was SF-900II. 35 201100545 The experimental results are the average of two independent cell culture experiments. Figure 4 shows the effect of the amount of oxygen in the IOSTAT® beta reactor on the yield of toxic particles. When the Sf 9 cells reached 1 xio ceiis/m, they were inoculated into the reactor and cultured in a 9-bath. The amount of dissolved oxygen in culture was controlled at 50%. When the cell concentration reached 4χ1〇6 cdls/mi after three days, the cells were infected with Bac-Pl-C3CD (MOI 1G), and the dissolved oxygen content after the money was -15, % or 60, respectively. % to compare the effect of different dissolved oxygen levels on the yield of alfalfa. The experimental positive control group (positive) with the same infection conditions (4 xi〇6ceus/m丨, Μ.] 1〇), but produced in the rotating angle bottle (undissolved oxygen) ugly virus county output n silk is __ The average of sub-independent cell culture CI experiments.

Figure 5 shows the type, size and surface of the virus. (a) and (8) transmission electron micrographs Kc) and (d) immunogold staining combined with transmission electron microscopy confirmed that the viral particles contained VP1 protein. The experimental samples were obtained by infecting Sf-9 cells with Bac-Pl-C3CD, purifying the obtained viroid-like particles (a and c), or infecting High_5 cells with Bac_P1_3CD, and purifying the virus-like particles (b and d). Bar, 100nm. CJ Figure 6. Anti-EV71 IgG antibody titer analysis induced by injection of viroid-like particles into mice. Negative control group PBS: sterilized PBS was mixed with an adjuvant (1:1) as an injection sample. Negative control group r-BV: Sf-9 was infected with r_BV without any foreign protein gene. Positive control grade EV71: Enterovirus 71 virus purified by sucrose discontinuous gradient centrifugation. Positive control group Bac-Pl-3CD: viroid-like particles produced by infecting Hi-5 cells with Bac-Pl-3CD. Experimental group Bac-Pl-C3CD: 36 201100545 virus-like particles produced by infecting Sf-9 cells with Bac-Pl-C3CD. This is inconsistent with the injection of humans, and on the 3rd week, the face is shot. Serum was collected at week 7 and sequenced (minimum dilution ratio of 23) and analyzed. Figure 7. Neutralization potency test of antibodies induced by virus-like particles for different enterovirus strains. (4) β for the enterovirus strain top 2272,98 (genGgiOup (2) towel and titer. (6) serum neutralization titer of enterovirus type 71 gen〇gr〇upB5 virus. Negative control group pBs: with sterilized PBS and adjuvant Mix (1:1) as the injection sample. Negative control group r_Bv: without any source protein base _ t_BV recorded Sf 9. Shirt group EV<71: 4 sugar discontinuous gradient, purified by the method Enterovirus 71 virus. Positive control group Bac_pi_3CD: viroid-like particles produced by infecting Hi_5 cells with Ba〇Pl-3CD. Experimental group Bac-Pl-C3CD: viroid-like particles produced by sf 9 cells infected with Bae_pi C3CD. Five groups of samples were mixed with adjuvant, injected into the peritoneal cavity of mice, and boosted in the fourth week. Serum was collected at week 7 and serially diluted (minimum dilution ratio was 23), and then analyzed. The minimum dilution ratio of serum is 23. [Main component symbol description] No 37

Claims (1)

201100545 VII. Scope of Patent Application·· L-type baculovirus transposition vector showing viral egg self-quality, comprising: a. a strong stem virus promoter and a nucleotide sequence linked downstream of the promoter, wherein The nucleotide sequence can be translated into a viral virus-white; and b. - a weak promoter and a - segment are linked to a stucate phase downstream of the promoter, wherein the nucleotide sequence can be translated into a viral shell protein of the hydrolyzed virus A hydrolyzed protein in which a strong baculovirus promoter drives a nuclear profiling sequence linked to a nuclear Wei sequence downstream of a cascade mover to produce a protein-producing protein that is translated by a nucleotide sequence linked downstream of the promoter. The baculovirus transposition vector according to claim 1, wherein the virus refers to enterovirus 0, such as the baculovirus transposition vector described in claim 2, and the enterovirus refers to enterovirus 71. type. 4. The baculovirus transposition vector of claim 1, wherein the strong baculovirus promoter is selected from the group consisting of a baculovirus polyhedrin promoter or a piG promoter. The baculovirus transposition vector of claim 1, wherein the weak promoter is selected from a baculovirus immediate early promoter, a cytomegalovirus promoter or other weak promoter that acts in a baculovirus. child. The baculovirus of the present invention is a recombinant baculovirus, which comprises a vector according to the scope of the patent application. A recombinant baculovirus as described in claim 6, which is for infecting a host cell to produce a recombinant protein. 38 6. 201100545 8. The heavy-duvirus as described in __7, the viral hull protein and the hydrolyzed protein of the virus. Go white matter refers to the intestine 9. - The type of the county egg _ is made of silk red _, _ the infection of the recombinant baculovirus described in Item 6. * Patent application scope, for example, the baculovirus host cell described in claim 9 of the patent application, Ο 目 Μ Η-9 or Ηι_5 cell line. A host cell according to claim 9, which further comprises the viral protein as a viroid-like particle of the virus and released to the outside of the cell. The composition of the white matter of the money, such as the 12th fine tear of the food, _, the virus shell protein and the hydrolyzed egg of the plum... The poisonous 14. A method for preparing the virus-like particle, the step of which is to construct the patent scope of the patent. The rod-shaped _ butterfly; (8) the lobes are secreted to form a wall rod wheel such as a shaft; and (7) the arsenic virus is infected to form a host cell as described in claim 9 of the patent application. The method of claim 2, further comprising, after culturing, centrifuging the mash to obtain viroid-like particles. 16. The method of claim 14, wherein the viroid-like particle is a virus-like particle of enterovirus. 17. A vaccine for the use of a simple anti-viral filament, which is selected from the group consisting of a recombinant rod-shaped scale as described in the application of claim 1, and the host cell and the host cell as described in (4) Patent No. 9. A group of released virus-like particles, and an immunological adjuvant acceptable for use in medicine 2011 201100545. 18. The vaccine of claim 17, wherein the virus is an enterovirus.