Preparation Of Monoclonal Antibody Against Canine Distemper Virus And Preliminary Development Of A Biosensor For Determing CDV

Canine distemper（CD）is a highly contagious animal disease caused by animal canine distemper virus（CDV）,which is widely distributed worldwide.It is highly infectious and has high incidence rate and mortality.It is listed as the two category of animal diseases in China and has become one of the most serious diseases that threaten the health of dogs.In recent years,it has been reported that CDV can infect non-human primates,which also indicates that CD may develop into zoonosis.At present,there is no effective treatment for CD.Early prevention and vaccination are effective means of prevention and control.The main detection technologies of canine distemper include:virus isolation and identification,immunological detection technology（ELISA,IFA,GICA）,molecular biological detection technology（PCR,lamp）and gene chip detection technology.Virus isolation and identification is an effective technique for the diagnosis of canine distemper,but it is not suitable for clinical diagnosis because of the strict requirements for test conditions and technicians.ELISA has high sensitivity,but poor repeatability and complex operation,t is only suitable for serum detection.Immunofluorescence assay（IFA）has good accuracy,but it has high requirements for detection conditions.If it is not operated properly,it is easy to produce nonspecific fluorescence and affect the experimental results.Colloidal gold immunochromatography（GICA）is easy to operate,but due to its limited sensitivity,it is only used as a clinical auxiliary detection method for canine distemper.PCR can achieve the purpose of detection by detecting virus nucleic acid,but the price of detection equipment is expensive,and the detection time is long.LAMP detection technology has the advantages of high sensitivity and suitable for clinical field detection,but the primer design is cumbersome and prone to nonspecific amplification.Gene chip technology can rapidly detect viral nucleic acid and protein,but the high cost limits the large-scale application of clinical detection.Therefore,it is of very important to seek a convenient,fast,sensitive and specific detection method that can be used in clinical practice for the prevention and control of canine distemper.At present,most of the detection technologies of canine distemper produce immune reaction with antigen and antibody.Due to the different quality of CDV antigen and antibody products produced in China,although the quality of foreign imports is guaranteed,the price is high.Therefore,the preparation of CDV antigen antibody with high titer,strong specificity and low cost can provide an excellent reagent for the rapid detection of canine distemper.Fiber Bragg grating biosensors（FBG）have the characteristics of corrosion resistance,high temperature resistance,low price,high sensitivity,good reusability,good specificity and no labeling.There is no report on the detection of canine distemper virus by fiber Bragg grating biosensor.In this study,the FBG biosensor was further increase its detection and applied to the detection of canine distemper.In this paper,the canine distemper virus nucleocapsid protein（CDV N）was prepared by prokaryotic expression system,and the CDV N recombinant protein was injected intraperitoneally into BALB/c mice by long-term immunization method,and the spleen cells of immunized mice were fused with myeloma cells by PEG method.and then CDV N monoclonal antibody was produced.Using hydrofluoric acid（HF）to etch the fiber core of the fiber Bragg grating,hydroxylation,silanization,coating of gold nanoshells（Au NS）,carboxylation,and activation of the carboxyl groups on the surface of the grating,the CDV N monoclonal antibody was immobilized on the surface of the fiber grating by（-NH-CO-）covalent bond,and it was blocked with nonfat milk powder.Through a series of biological functional modifications,including grating hydroxylation,silylation,gold nano shell modification,11-MUA carboxylation and activation of hydroxyl groups,immobilization of CDV n monoclonal antibody and other steps,the FBG biosensor detection platform was established,and the sensor was evaluated,mainly including sensitivity,repeatability and specificity analysis,and the clinical samples were detected.In this paper,a rapid detection technology of canine distemper based on gold nano shell modified corrosion Bragg grating biosensor is preliminarily established,which provides a new detection method for the diagnosis of canine distemper.The research contents of this paper are as follows:1.According to the CDV N（accession number:DQ522030.1）gene sequence published on Gen Bank website,analyze and optimize it to make it suitable for E.coli expression system,chemically synthesize the whole CDV N gene,connect it with vector p ET28a（+）,and construct p ET28a（+）-CDV N recombinant plasmid.The recombinant plasmid was transformed into E.coli competent cells BL21（DE3）to carry out protein expression,and the protein induction conditions（time,temperature,IPTG concentration）were screened.According to the best induction conditions,the recombinant protein of CDV N was expressed in large quantities.The recombinant CDV protein was purified by affinity chromatography nickel column.The purity was identified by SDS-PAGE gel electrophoresis,the concentration was determined by BCA kit,and the specificity was identified by Western-blot.The immune reactivity of canine distemper antigen rapid test strip was identified.2.The prepared CDV N recombinant protein was fully emulsified,and BALB/c mice were injected intraperitoneally.After four times of immunization,the serum titer was measured.Scrape the mouse spleen that has been immunized for four times and one time of enhanced immunization and meets the fusion standard into a cell suspension,and fuse it with mouse myeloma cells（SP2/0）to obtain hybridoma cells,which are screened by indirect ELISA,The supernatant of cell fusion pore was screened,and the screened positive hybridoma cells were subcloned and screened twice to obtain monoclonal hybridoma cell lines that can stably secrete antibodies,and the titer was determined.The monoclonal cell lines were expanded and inoculated intraperitoneally into BALB/c mice to induce the production of ascites monoclonal antibody.The ascites was purified by protein a column,the antibody subtype was detected by sigma subtype identification kit,and the biological characteristics of the purified monoclonal antibody were identified.3.Soak the gate region of the fiber Bragg grating in 5%NHO₃to remove impurities.The FBG was etched with HF until the core diameter was about 6μm,and the surface of the grating was covered with hydroxyl groups（-OH）for hydroxylation with Na OH solution;Then,the grating is immersed in a silane coupling agent solution to bond trans to achieve surface silanization by covalent bonding.;Gold nanoshells（Au NS）decorate the surface of the grating,carboxylated the fiber grating with 11-MUA ethanol solution,activated the carboxyl group on the grating surface with EDC/NHS activator,and combined the prepared CDV n monoclonal antibody with the grating surface,and the prepared CDV N monoclonal antibody is combined with the grating surface to complete the modification of the FBG grating and build the FBG biosensor platform.Different concentrations of CDV N antigen were respectively tested for sensitivity,and the same concentration was determined three times for repeated experiments,and specific experiments were carried out for different viruses（avian influenza virus,Newcastle disease virus,rabies virus,canine distemper CDV N antigen）.Clinical experiments were carried out on several groups of canine distemper positive and negative clinical samples.result:1.The recombinant plasmid p ET28a-CDV N was identified by double digestion with NdeⅠand XhoⅠ,and a band appeared at 1581 bp,which was in line with expectations;When the induction temperature of p ET28a-CDV N/BL21（DE3）glycerol bacteria was 30℃,the final concentration of IPTG was 0.4 mmol/L,and the induction time was 8 h,the expression of CDV N recombinant protein was the highest.The recombinant gene engineering bacteria expressed a large number of proteins according to the best conditions,and purified them by affinity chromatography nickel column.The results of SDS-PAGE electrophoresis showed that the purity of CDV N recombinant protein was higher than 90%;the concentration of CDV N recombinant protein measured by BCA method reached 1.783 mg/m L;Western blot identification results can be seen at 60 KD obvious Western blot;Canine Distemper Antigen Rapid Test Card can detect clear bands.This indicated that the CDV N recombinant protein was successfully expressed in E.coli BL21（DE3）competent cells,and the immunoreactivity was good.2.Take the mouse spleen that has been immunized for 4 times of long-term and 1 time of booster immunization and reach the fusion standard,scrape it into a suspension,and fuse it with mouse myeloma cells（SP2/0）to prepare hybridoma cells,and the cell fusion rate is 100%,the positive rate was 93.8%.After two rounds of positive hybridoma cell screening and two subcloning screening,a total of 6 monoclonal cell lines were obtained,named:3-3G,1-10B,3-5A,1-9G,3-8H,5-8E.After cryopreservation,recovery and multiple passages,the cell line can still secrete high titer antibodies.After the identification of monoclonal antibody subtypes,the two hybridoma cells 3-3G and 1-10B were both of Ig G2 type,and the two monoclonal cell lines were expanded and cultured.The two monoclonal cell lines were expanded and cultured,injected into the peritoneal cavity of BABL/c mice to prepare ascites,and the ascites was purified by affinity chromatography through a Protein A column.SDS-PAGE gel electrophoresis identification showed that the monoclonal antibody had obvious bands at 25 KD and 50 KD,and the purity was good,and there was no obvious impurity band;the concentration of the antibody measured by BCA method was at least 2.78mg/m L;By Western blot identification,the prepared CDV N monoclonal antibody and purified CDV N antigen had a western blotting band at the expected 60kd,and the monoclonal antibody titer reached 1:2048000.The CDV N monoclonal antibody prepared in this study lays the foundation for the subsequent research on the detection method of canine distemper.3.The FBG detection platform and the CDV N antibody detection method was established.The FBG fiber grating biosensor was successively subjected to hydroxylation,silanization,modification of gold nanoshells,activation,immobilization of canine distemper monoclonal antibody and blocking of skim milk powder.With the modification of the fiber surface in each step,its amplitude gradually decreased,and its total amplitude decreased by 0.542d B after the functional modification.After coating with gold nanoparticles,the amplitude change is the largest,which is 0.357d B lower than the previous step,accounting for 69.1%of the overall change.The detection limit of the biosensor for CDV N antigen was in the range of 50 pg-10μg/m L,and the saturation concentration was about～10μg/m L.Through three repeated tests,the results of amplitude difference show that the difference is small and the repeatability is good.The specificity of the sensor was verified by detecting avian influenza virus,Newcastle disease virus,rabies virus,and canine distemper CDV N antigen,and the results showed that avian influenza virus,Newcastle disease virus and rabies virus did not react with CDV N monoclonal antibody on the sensor,but reacted strongly with CDV N antigen of canine distemper,indicating that the FBG sensor had good specificity.;through the detection of positive and negative samples of canine distemper,the results show that only a few groups of positive clinical samples have large amplitude intensity changes,indicating that the sensor can meet the requirements of clinical detection and has a good prospect of clinical application..In conclusion:In this study,p ET28a（+）-CDV N/BL21（DE3）genetically engineered bacteria were successfully constructed,and the CDV N recombinant protein was successfully expressed in prokaryotic expression system.Using the recombinant protein as an immunogen,mice were inoculated by long-term immunization.Six cell lines secreting CDV N monoclonal antibody were prepared by PEG cell fusion,subcloning and screening.Using the prepared CDV N antigen and monoclonal antibody,a rapid detection method for canine distemper virus based on gold nanoshell-modified corrosion Bragg biosensor was established.It was evaluated in terms of sensitivity,reproducibility,specificity,and detection of clinical samples.The results show that this method has the advantages of high sensitivity,strong specificity,good reproducibility,and low price,and can be used for the detection of CDV N in clinical samples of canine distemper,providing a new detection technology for the early and rapid diagnosis of canine distemper virus.