| Vaccines play an extremely important role in the prevention and control of human and animal diseases.According to the continuous progress and development of vaccine research,vaccines can be divided into traditional inactivated,attenuated vaccines and a variety of new vaccines under development.Nanoparticles can be used as a delivery system.The nanoparticles structure vaccine can wrap the antigen and prevent the antigen from being digested and degraded prematurely,thus obtaining a high immune response.The nano-materials used in nanoparticles,such as ferritin and virus-like particles,are obtained from nature,and the naturally obtained biological nanoparticles have good safety and stability.The research content of the first polyprotein nanoantigen in this paper is mainly divided into two chapters:The first chapter is the exploratory study of the foot-and-mouth disease polyprotein nanoantigen.In this chapter,based on the long-term epidemic subtypes of foot-and-mouth disease virus type O,type A and Asia 1,the corresponding nanoparticle structure vaccine of foot-and-mouth disease was developed.His/HE/EW29 was used as an affinity tag to construct and prokaryotic expression with 9 lysolytic tags Grifin,GST,MBP,Sumo,Thioredoxin,y-crystallin,ArsC,PpiB,CeHSP17 and three foot-and-mouth disease antigenic proteins SeFnt16599,CcFnt166AS and SeFntl 6798.The optimized FMDV VP1 protein and ferritin were concatenated.81 engineering strains were induced to express and purified by affinity chromatography,and the target proteins with enhanced solubility were obtained.compared with other solubilizing tags,chimeric MBP tags effectively promoted the expression and solubility of target proteins.Finally,the engineering strain containing His-MBP-SeFnt16599/CcFnt166AS/SeFnt16798(FMDV VP1 protein and ferritin tandem protein)recombinant vector was selected as the target strain for follow-up test.The purified protein was self-assembled under the condition of pH value 8.0.The Western blot results showed that the recombinant protein could be specifically recognized by guinea pig anti-FMDV high immune serum and had good immune activity.In order to detect the antibody level and antibody titer of soluble recombinant foot-and-mouth disease antigen protein His-MBP-SeFnt16599/CcFnt166AS/SeFnt16798,we established an animal model of guinea pig.The recombinant protein and Freund's complete/incomplete adjuvant were emulsified and injected into guinea pigs.Two weeks and four weeks later,enhanced immunity was carried out.Blood samples were collected and sera were collected on the 42 nd day,and the antibodies were analyzed by SeFnt16599/CcFnt166AS/SeFnt16798 test group.The specific antibody against antigen could be detected 14 days after immunization.with the extension of immunization time,the level of serum antibody continued to increase,and the level of antibody increased significantly after enhanced immunization.The purified His-MBP-SeFnt16599/CcFnt166AS/SeFnt16798 was used as coating antigen,the last immunized guinea pig serum was used as negative control,and the blank control group was set up.The antibody titer in guinea pig serum was detected according to the routine indirect ELISA operation procedure.The results showed that the last antibody titer reached 1ELISA 204800,indicating that His-MBP-SeFnt16599/CcFnt166AS/SeFnt16798 protein has good immunogenicity and can induce specific immune response.The experiment in this chapter provides a new idea for the prevention and control of foot-and-mouth disease,a new way for the development and application of a new structural vaccine of foot-and-mouth disease,and lays an experimental foundation for the follow-up research of nanoparticles structure vaccine of different subtypes of foot-and-mouth disease virus.The second chapter is the exploratory study of the polyprotein nanoantigen of the universal epitope of coronavirus.The purpose of this study is to construct a phage Q?capsid protein chimeric virus-like particle(virus-like particles,VLPs)vaccine with common antigenic epitopes of coronavirus,which is also a kind of nanoparticle structure vaccine.The Q? capsid protein of RNA phage was designed and synthesized and recorded as Qbeta;.The common antigen epitope CoV epitope of coronavirus was inserted into the C-terminal of Qbeta by gene recombination,and Qbeta-CoV;was inserted into the N-terminal of Qbeta-CoV,followed by TEV restriction site,mCherry red fluorescent tag and modified affinity purified histidine tag 6xHE,which was recorded as HE-Qbeta-CoV.In BL21(DE3),the expression of Qbeta,Qbeta-CoV and HE-Qbeta-CoV was induced by IPTG.The fusion protein was purified by ammonium sulfate salting-out and chelate metal affinity chromatography and the particle morphology was observed by electron microscope.The sera of mice immunized with Qbeta-CoV and HE-Qbeta-CoV chimeric VLPs vaccines were collected,and the antibody level and titer were detected by ELISA.The results showed that the fusion proteins Qbeta,Qbeta-CoV and HE-Qbeta-CoV were successfully expressed,and high purity particles were obtained after purification.The morphology of Qbeta-CoV and HE-Qbeta-CoV chimeric particles was similar to that of Qbeta particles,forming chimeric VLPs;.In addition,two kinds of chimeric VLPs vaccines induced specific antibody response in mice.In this chapter,the chimeric VLPs,of phage Q? capsid protein with universal epitopes of coronavirus was successfully constructed,which provides a potential vaccine for the treatment of coronavirus disease.In summary,different subtypes of foot-and-mouth disease virus nanoparticle structure antigen protein and coronavirus common epitope nanoparticle structure antigen protein were obtained by prokaryotic expression.It has laid the experimental foundation for the development and application of related vaccines,and the advantage of nanoparticles is expected to become a new technological route to promote the technological progress of vaccine development.When foreign invading bacteria,viruses,fungi and other foreign substances stimulate the body,the immunoglobulins secreted by B lymphocytes to resist stimulation are called antibodies.The molecular interaction of specific binding between antigen and antibody has important research value in many fields such as biology.Antibodies play an important role in the diagnosis,detection and treatment of animal diseases.The polyclonal antibodies obtained from the serum by immunizing experimental animals are not obtained against a single antigen;the specific monoclonal antibodies obtained by B-lymphocyte hybridoma technology have always been the focus of related research,such as disease treatment and immune drug research;and the discovery of nano-antibodies in camels and other animals has brought great impetus to the development of science and technology.The second part of this thesis is mainly divided into three chapters:The first chapter is the prokaryotic expression and functional verification of the fusion protein of ferritin and nano-antibody against porcine epidemic diarrhea virus membrane protein.The purpose of this study was to prepare the highly soluble fusion protein Fenobody-PEDV,of recombinant ferritin and nano-antibody against porcine epidemic diarrhea virus membrane protein,and to explore the application of fusion protein Fenobody-PEDV in the diagnosis and treatment of PEDV.The nano-antibody gene of ferritin ferritin gene and anti-PEDV membrane protein was designed and synthesized,which was named Fenobody-PEDV,and ligated to the vector pET21b.After the construction of the vector,the fusion protein was expressed in E.coli expression system.The single fusion protein Fenobody-PEDV,with the size of 33.88 kDa was purified by ammonium sulfate salting-out and examined by electron microscope.The prepared recombinant protein Fenobody-PEDV was labeled with HRP and used as direct ELISA test and virus neutralization test with PEDV strain.The results showed that the fusion protein Fenobody-PEDV;with high purity was obtained after prokaryotic expression and purification,and the electron microscope results showed that the recombinant protein Fenobody-PEDV self-assembled into nanoparticles with a diameter of 20 nm.The results of direct ELISA showed that the recombinant protein Fenobody-PEDV could bind to the specific antigen FEDV strain at a certain concentration and had high activity.Under the condition of a certain concentration of recombinant protein Fenobody-PEDV,it can sensitively recognize specific antigens in the range from low concentration to high concentration;virus neutralization test showed that recombinant protein Fenobody-PEDV had certain neutralizing activity to PEDV.In this experiment,a stable method for obtaining recombinant protein Fenobody-PEDV was established,and its application in direct ELISA was explored,and it was proved that it had certain neutralizing activity.The successful preparation of the recombinant protein Fenobody-PEDV,laid the experimental foundation for the further study of the fusion protein of ferritin and nano-antibody against porcine epidemic diarrhea virus membrane protein in the diagnosis,treatment and follow-up research of PEDV.In the second chapter,the expression and functional verification of nano-antibodies against porcine pseudorabies virus gE protein.The aim of this study was to express soluble anti-porcine pseudorabies virus gE protein nano-antibodies in prokaryotic cells,and to explore the application of anti-porcine pseudorabies virus gE protein nano-antibodies in the diagnosis and treatment of porcine pseudorabies virus.The gene sequence of nano-antibody against porcine pseudorabies gE protein was designed and synthesized,which was named PRVgENb;.The fusion protein was expressed in E.coli expression system after the construction of PRVgENb and pET21b vector.After purification by Ni2+,a single nano-antibody PRVgENb;with the size of 16.39 kDa was obtained.The prepared nano-antibody PRVgENb was labeled by HRP,and the PRV strain was used for direct ELISA test and virus neutralization test.The results showed that the fusion protein PRVgENb;direct ELISA with high purity was obtained after prokaryotic expression and purification.The results showed that under the condition of certain antigen concentration,the nano-antibody PRVgENb could bind to the specific antigen at lower concentration and had higher activity,while under the condition of certain antibody concentration,the nano-antibody PRVgENb could sensitively recognize the specific antigen in the range from low concentration to high concentration.The results of virus neutralization test showed that nano-antibody PRVgENb could inhibit the replication of PRV and had certain neutralizing activity.In this experiment,a stable method for obtaining nano-antibody PRVgENb was established,and its application in direct ELISA was explored,and it was proved that it could inhibit the replication of PRV.Nano-antibody PRVgENb lays an experimental foundation for the diagnosis,treatment and follow-up research of PRV.The third chapter is the prokaryotic expression and functional verification of nanobodies against vesicular stomatitis virus nucleoprotein and polyantinucleoprotein nanobodies.The purpose of this chapter is to prepare highly soluble anti-vesicular stomatitis virus nucleoprotein nanobody VSVNb and recombinant truncated ferritin and anti-vesicular stomatitis virus nucleoprotein nanobody fusion protein FnL-VSV.The application of VSVNb and FnL-VSV in the diagnosis of VSV was initially explored.Obtain the anti-vesicular stomatitis virus nucleoprotein VHH sequence from the alpaca and named it VSVNb;after truncating the ferritin isolated from Archaea thermophilus,and obtain the anti-vesicular stomatitis virus nucleoprotein from the alpaca The VHH sequence is connected in series and named FnL-VSV.After constructing the vector,the E.coli expression system was used for prokaryotic expression of the fusion protein.After Ni2+ purification,a single nanobody VSVNb with a size of 15.95 kDa was obtained;a single fusion protein FnL-VSV with a size of 29.04 kDa was obtained after purification by ammonium sulfate salting out,and Perform electron microscopy testing.The prepared recombinant proteins VSVNb and FnL-VSV were respectively labeled with HRP,and used for direct ELISA test with the VSV strain.The results showed that the fusion proteins VSVNb and FnL-VSV with higher purity were obtained after prokaryotic expression and purification;electron microscopy results showed that the recombinant protein FnL-VSV self-assembled to form nano-particles with a diameter of 20 nm;the direct ELISA results showed that in the antigen Under certain concentration conditions,the recombinant proteins VSVNb and FnL-VSV both bind to the specific antigen VSV strain under lower concentration conditions and have higher activity,and the recombinant protein FnL-VSV has a larger OD450nm value than VSVNb;Under certain conditions of protein VSVNb and FnL-VSV concentration,it can sensitively recognize specific antigens in the range of low concentration to high concentration.In this experiment,a method for stably obtaining recombinant protein VSVNb and FnL-VSV recombinant protein was established,and its use in direct ELISA was preliminarily explored.The recombinant proteins VSVNb and FnL-VSV were successfully prepared,which laid the experimental foundation for further research on the diagnosis of Nanobodies in VSV and the follow-up research on antiviral drugs.In summary,this experiment initially obtained ferritin and anti-porcine epidemic diarrhea virus membrane protein nanobody fusion protein,anti-porcine pseudorabies virus gE protein nanobody,nanobody against vesicular stomatitis virus nucleoprotein and Truncated ferritin nanobody.The simple,effective,and large-scale acquisition of Nanobodies has laid the experimental foundation for the development and subsequent applications of various virus-related antibodies.Secondary antibodies have very important application significance in immunoassay techniques such as WB,IFA,and ELISA.The traditional method of obtaining polyclonal anti-mouse IgG and anti-rabbit IgG secondary antibodies requires the use of a large number of animals,and the antibodies require complex in vitro labeling.The nanobody sequence obtained from camel animals can be used to produce anti-mouse IgG and anti-rabbit IgG secondary nanobodies through Escherichia coli,which can effectively avoid the problem of using animals and greatly reduce production costs.At present,the choice of antibody labeling enzyme is mainly HRP with high sensitivity.The Ca2+binding site and disulfide bond contained in HRP make it impossible to express correctly in the prokaryotic expression system,and it can be expressed in low Ca2+concentration and reducing environment(such as cytoplasm).)Does not function properly.A newly discovered bacterial alkaline phosphatase has higher activity and stability.At the same time,there is a targeted modification of ascorbate peroxidase that can be expressed and produced in almost all cell structures.Both of these enzymes can It is well expressed and applied in the E.coli prokaryotic expression system and is expected to become a substitute for traditional HRP.In addition,the design of serial expression of enzyme and nanobody in the prokaryotic expression system reduces the complicated steps of in vitro labeling,and has obvious application advantages.The third part of this thesis is the preliminary exploration of the application of multimeric protease-labeled nanobodies in immunoassay technology.The main content of the research is the prokaryotic expression of enzyme-labeled anti-mouse IgG nanobodies and their preliminary application in new self-made ELISA.The anti-mouse IgG nanobody isolated from the alpaca and the modified alkaline phosphatase AP1 gene sequence were serially named MGNb-AP1,and the anti-mouse IgG nanobody isolated from the alpaca and the modified ascorbic acid peroxidation The sequence of the AP2 gene of the enzyme is named MGNb-AP2 in tandem,and the sequence of the anti-mouse IgG nanoantibody isolated from alpaca and the ALFA-tag gene sequence of a universal epitope tag is tandemly named MGNb-ALFA-tag,which will be named MGNb-ALFA-tag.The anti-mouse IgG nanobody,the epitope tag ALFA-tag,and the truncated ferritin gene sequence isolated from the alpaca were tandemly named FnL-MGNb-ALFA-tag.The tag Nanobody and the modified alkaline phosphatase AP1 gene sequence were concatenated and named NbALFA-AP1.The ALFA-tag Nanobody isolated from the alpaca and the modified ascorbate peroxidase AP2 gene sequence were concatenated Named NbALFA-AP2.The gene sequences were optimized by E.coli codons.After gene synthesis,they were cloned into the vector pET21b.The E.coli expression system was used for the prokaryotic expression of the recombinant protein.After Ni2+purification and urea denaturation and renaturation,the nano antibodies MGNb-AP1 and MGNb-were obtained.AP2,MGNb-ALFA-tag,FnL-MGNb-ALFA-tag,NbALFA-AP1 and NbALFA-AP2 fusion proteins.The prepared Nanobodies were sequentially added to perform new ELISA detection according to the three combinations of one-step detection method,two-step detection method and high-sensitivity two-step detection method set in the experiment.The results showed that the recombinant target proteins MGNb-AP1,MGNb-AP2,MGNb-ALFA-tag,FnL-MGNb-ALFA-tag,NbALFA-AP1 and NbALFA-AP2 were obtained after prokaryotic expression and purification;the ELISA results showed that the one-step detection method,Both the two-step detection method and the high-sensitivity two-step detection method can qualitatively detect the mouse antibody to be tested.This experiment established a method for stably obtaining a series of enzyme-labeled anti-mouse IgG nanoantibodies MGNb-AP1,MGNb-AP2,MGNb-ALFA-tag,FnL-MGNb-ALFA-tag,NbALFA-AP1 and NbALFA-AP2 recombinant proteins.And preliminarily explored the application of a series of enzyme-labeled nanobodies in new ELISA detection.A series of enzyme-labeled nano-antibodies have laid the experimental foundation for the follow-up research of the new ELISA detection system,and provided a new idea of technological innovation for the ELISA detection system,which is expected to become a new ELISA detection technology route. |
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