| Rabies is a zoonotic infectious disease characterized by acute,progressive and fatal encephalomyelitis caused by rabies virus(RV).Rabies is one of the infectious diseases with the highest fatality rate.Once neurological symptoms appear,the fatality rate is almost 100%.my country is seriously endangered by rabies.The number of reported cases is only lower than that of India,ranking second in the world,and the death toll from rabies ranks among the top three in deaths from various infectious diseases.Rabies has seriously affected my country's public health security.At present,there is no effective treatment for rabies.Early prevention and vaccination are effective prevention and control methods.Therefore,early diagnosis of rabies is particularly important.At present,the main detection technologies for rabies include virus isolation and identification,fluorescent antibody technology,enzyme-linked immunosorbent assay(ELISA),polymerase chain reaction(PCR),dot enzyme immunoassay(DIA)and colloidal gold.The isolation,identification and diagnosis of the virus is relatively accurate,but it takes a long time,and the operation is cumbersome,which is not conducive to rapid detection;fluorescein-labeled antibodies can be used for antigen localization.The degree and research experience have a great influence,and it is not suitable for spoiled samples and samples preserved in formalin;ELISA method is simple to operate,suitable for large-scale serological investigation,and has great application prospects,but requires repeated washing,and the detection sensitivity is limited,and requires a certain number of samples;although the PCR method can directly detect the gene of the virus and has high detection sensitivity,but the detection cost is high,easy to contaminate,and the technical requirements are relatively high,which is only suitable for laboratory diagnosis,not suitable for rapid diagnosis of clinical infection;DIA method has the advantages of rapidity,sensitivity and specificity,but due to the influence of non-specific adsorption,diffusion limitation and high-dose hook effect,it can cause special effects of "false low value" or even "false negative" false results;conventional colloidal gold method At present,there are related products at home and abroad,but their detection sensitivity is not high,the product quality varies greatly,and it is not suitable for quality control.Therefore,the establishment of a fast,simple,sensitive and low-cost detection technology is of great significance for the prevention and control of rabies.The existing rabies serological detection methods are mainly based on the specific reaction of antigen and antibody,but the quality of domestic rabies virus antigen and antibody is uneven,and there are mature commercial antibodies abroad,but the price is very expensive.Therefore,the preparation of rabies-specific antigens and antibodies with high titer,strong specificity and low cost can provide effective reagents for early diagnosis of rabies.Micro-nano optical fiber biosensors have the advantages of high sensitivity,good specificity and fast response speed for detection due to their strong confinement ability,strong evanescent field,abnormal waveguide dispersion,and good mechanical strength and bending performance.In this paper,on the basis of the previously established optical fiber sensor,a micro-nano optical fiber biosensor is constructed and improved to improve its detection sensitivity,stability and specificity,and it is applied to the detection of rabies antibodies.In this paper,rabies nucleocapsid protein(NP)was prepared by prokaryotic expression system,the prepared NP protein was immunized in BALB/c mice,and NP monoclonal antibody was prepared by PEG1500 fusion technology,which provided an effective reagent for the diagnosis of rabies.A micro-nano optical fiber sensor platform was established,and the initial refractive index of the optical fiber was calibrated,the surface of the optical fiber was pretreated,and the surface of the optical fiber was hydroxylated,silanized,and coated with graphene oxide(GO)and immobilized the NP antigen on the surface of the optical fiber,and then sealed it with nonfat milk powder for 1 h to detect rabies antibodies at different concentrations.Finally,the rabies antibody micro-nano optical fiber biosensor was evaluated(including detection sensitivity,repeatability,specificity),and the clinical specimens were tested and analyzed,so as to establish a new detection method for the diagnosis of rabies.The research contents of this paper are as follows:(1)According to the RV NP gene sequence corresponding to the NCBI database(Gen Bank accession number: 1489853),the gene was codon-optimized,the RV NP gene was chemically synthesized,and cloned into the prokaryotic expression vector p ET28a(+)to construct a recombinant The plasmid p ET28a(+)/RV NP was transformed into E.coli BL21(DE3)competent cells for induction expression,and the induction time,temperature and IPTG concentration were screened,and the RV NP protein was tested according to the best induction conditions.A large amount of expression was carried out,and the His-tag nickel column was used for purification.The purified RV NP recombinant protein was subjected to purity analysis by SDS-PAGE electrophoresis,concentration determination by BCA protein quantitative kit,and specific identification by Western-blot.Immunoreactivity assays were performed with commercially available RV antibodies.(2)The RV N recombinant protein purified by nickel column was mixed and emulsified with Freund's adjuvant in equal proportions,and BALB/c mice were immunized with 100 ug each,with an interval of two weeks each time,and the assay was performed after four immunizations.The antibody titer of mouse serum was obtained,and the spleen of the mouse was taken to make a cell suspension and fused with myeloma cells.The supernatant of the fused hybridoma cells was screened by ELISA method,and the hybridoma cells with higher positive value were selected for subcloning by limiting dilution method.After 3 rounds of subcloning,the obtained monoclonal hybridoma cell line was expanded.Cultivated,induced antibody production by intraperitoneal injection of mice,and carried out biological characterization of monoclonal antibodies,including subtype identification,SDS-PAGE purity identification,ELISA titer determination,Western-blot specificity identification,and BCA concentration determination.(3)Before the micro-nano fiber is not treated,its refractive index is calibrated to obtain its consistency test(RI)sensitivity value.The surface of the fiber was pretreated with 5% dilute nitric acid(HNO3),and graphene oxide(GO)was combined with the micro-nano fiber by electrostatic bonding.Then soak the micro-nano fiber with sodium hydroxide(Na OH)solution to hydroxylate and expose the surface carboxyl groups,and record the spectral data of the sensor during the modification process.The surface of the micro-nano fiber was silanized by covalent bonding with 3-aminopropyltriethoxysilane(APTES),and then GO was coated on the surface of the fiber,and carbodiimide(EDC)and N-Hydroxythiosuccinimide(NHS)activates-COOH on the GO surface.The RV NP antigen was immobilized on the surface of the micro-nano fiber by covalent bonding.300 u L of 5% nonfat dry milk was used for blocking treatment,and then the RV NP antibodies at different dilution concentrations were detected.After that,the repeatability,specificity and clinical performance of the sensor were evaluated and compared with other detection methods.result:(1)After double-enzyme digestion of the recombinant plasmid p ET28a(+)/RV NP,the target band appeared at 1359 bp,which was completely consistent with its corresponding known gene sequence after sequencing,indicating that the recombinant plasmid was successfully constructed.Will(2)The plasmid was transformed into Escherichia coli BL21(DE3)competent cells to screen for the best inducible expression conditions.It was identified by SDS-PAGE that the RV NP recombinant protein had the highest protein expression when induced for 6 h at 30°C and 0.1mmo L/L IPTG concentration.The protein concentration was 0.9239 mg/m L,and Western blot analysis showed that there was an obvious specific band at 51 KD.Afterwards,it was identified by ELISA reaction with RV NP monoclonal antibody purchased from Luoyang Gushuo Biotechnology Co.,Ltd.When diluted to 1:1024000 by the concentration of 6?g/m L and the antibody of 4mg/m L,the antigen-antibody still had obvious reaction,indicating that the prepared RV NP had good immunoreactivity.(2)In the RV NP monoclonal antibody preparation experiment,a total of two cell fusions were performed,the first fusion rate was 52.08%,and the positive rate was2.08%.The second fusion rate was 82.5%,and the positive rate was 72.9%.After three subcloning,a total of 4 monoclonal hybridoma cell lines that stably secreted antibodies were obtained,which were named 1F1-4E-8C,1F1-4C-2B,3C2-3F-8G,3F7-9G-2D,the subtype identification kit showed that they were all Ig G1 type.Two cell lines,1F1-4C-2B and 3C2-3F-8G,were selected to expand the culture and prepare mouse ascites.The ascites was purified with Protein A column,and the elution peaks were collected and identified by SDS-PAGE.The results showed that there were obvious lines at 25 KD and 50 KD.The band is consistent with the heavy chain and light chain bands of Ig G-type antibodies.After concentration determination by BCA method,the concentration of purified ascites monoclonal antibody was higher,reaching 0.9684 mg/m L.The ELISA results showed that the titers of 1F1-4C-2B and3C2-3F-8G ascites monoclonal antibodies could reach 1:64000.The four hybridoma cell lines could secrete high titer antibodies after repeated cryopreservation,resuscitation and multiple passages.,The results of Western-blot identification showed a strong reaction with RV NP antigen,and the specificity was good.(3)Established a rabies micro-nano optical fiber biosensor detection platform.The micro-nano fiber was successively subjected to refractive index sensitivity calibration,dilute HNO3 treatment,hydroxylation,silanization,coating with GO,activation of carboxyl groups,modification of RV N antigen,and detection after blocking.The experimental results show that the wavelength red shift of each step is:0 nm,8.754 nm,24.230 nm,30.032 nm,33.464 nm,34.064 nm,indicating that each modification process is ideal.The experimental results show that the detection limit of the sensor is 200 fg/m L,and the detection sensitivity is 200 fg/m L-10 ng/m L.The samples were repeatedly tested for several times,and the test results were consistent,indicating that the repeatability was good.Antibodies against other pathogens,such as Brucella outer membrane protein OMP16 antibody,canine distemper virus nucleocapsid protein antibody,H9N2 avian The specificity experiment of the influenza hemagglutinin protein antibody was carried out,and the results showed that the wavelength peak of the sensor did not change basically,and did not react with other antibodies,indicating that the specificity was good.Finally,the clinical serum samples of rabies were measured,and it was found that the clinical samples were obviously combined with the antigens on the sensor,indicating that the sensor can be used for the detection of clinical samples.Conclusion: In this study,p ET28a(+)/RV NP/BL21(DE3)genetically engineered bacteria were successfully constructed,and the RV NP protein was efficiently expressed in the prokaryotic expression system.Subcloning,and finally obtained 4cell lines that can stably secrete RV NP antibody.Using the developed RV NP antigens and antibodies,a method for rapid detection of rabies antibodies based on micro-nano optical fiber biosensors was established.The sensitivity,reproducibility,specificity,and clinical application of the device were analyzed and compared with other methods.The method has the advantages of high sensitivity,strong specificity,good repeatability,etc.,and it can be used for the detection of RV antibodies in samples,and provides a new detection technology and means for the diagnosis of rabies.In addition,the highly active and specific antigen and antibody reagents prepared in this study can provide effective reagents for the prevention and control of rabies... |
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