Development And Application Of Human Rabies Virus Nucleoprotein, Glycoprotein Quantitative Detection Reagents

Background and PurposeRabies Virus (RV) belongs to Lyssavirus, Rhabdoviridae, bullet-shaped,180nm long,75nm diameter, its head is hemispherical, it gets flat end. The virus particles are composed by two parts of the shell and core. Its genome are consisted of11928to11932nucleotides, containing five large open reading frame as a single chain, non-segmented negative-strand RNA, to encode five structural proteins. The arrangement sequence from the3’end to the5’end of genome are N, P, M, G and L genes, respectively encoding:virus nucleoprotein (NP), phosphoprotein (NS), Matrix Scientific protein (MP), glycoprotein (GP) and polymerase (LP).Recent studies show that the immune response of neutralizing antibodies, produced by rabies virus vaccine induces body, is mainly related to nucleoprotein and glycoprotein.Virus particles contain a large number of Nucleoprotein. Nucleoprotein accounts for36%of the total protein of the virus. It is the most stable protein of the virus, Its gene sequence is relatively constant, less point mutation,98%-99.6% homology of different strains. Nucleoprotein can be used as detection antigen, and is the main basis of parting Rabies Virus. It can induce protective immunity, is a king of T helper cell (Th) antigen that is able to induce cross-protection of different rabies virus, protein region21～35, region394～408, region404～418is primarily a Th cell epitope. After Rabies vaccination, human peripheral blood isolated from the rabies-specific reactive T cells are more than other after vaccination the number of specific T cells isolated. In these specific reactive T cells, most are Th cells, they are substantially all the majority of performance of the nucleoprotein specific reaction, suggesting that the main component of the nucleoprotein induced rabies cellular immune. In addition, the nucleoprotein can also to promote the generation of neutralizing antibodies. Early NP Freund’s complete adjuvant-free mice, strengthen inactivated rabies virus neutralizing antibody levels increased significantly, while GP strengthen, in obvious and antibody production. NP antibody can inhibit the propagation of the propagation of rabies virus in the cell between and within cells. Therefore, nucleoprotein is one of the indispensable ingredient of the rabies virus vaccine components, is an important indicator of vaccine vitality assessing.GP is the only antigen that is able to stimulate the body to produce neutralizing antibodies, and the holding of its antigenicity mainly depends on its spatial conformation. Play an important role in the process of the structural characteristics of the rabies virus GP is closely related to. its antigenicity and immunogenicity, which stimulate the body’s immune response. GP is the major protective antigen of the rabies virus. B, and T-cell recognition sites, capable of inducing the body from the cellular and humoral immune response to stimulate the body’s secretion and antibody. Therefore, the carboxy terminus of glycoprotein is missing its main antigenic sites point spatial conformation are broken or missing is the Another key indicator to measure the the rabies vaccine antigens vitality good or bad. The rabies virus nucleoprotein, glycoprotein content are the main quality indicators of the rabies vaccine, in the process of refining rabies vaccine often need timely detection of the antigen content, has been with the NIH animal Determination all these years. Long cycle, complex operation, as well as the constraints of the International Association for the Protection of Animals in experimental animals, does not apply to the monitoring of the various steps in the purification process. Therefore, the search for reliable in vitro detection of rabies virus vaccine efficacy of experimental methods to replace the NIH method is imperative.In this study, using time-resolved immunofluorescence analysis (TRFIA) technology developed rabies virus N protein, G protein quantitative detection reagents. The assess of the performance indicators, and comparison with other detection kit, to evaluate the feasibility of clinical detection applications. The time-resolved fluoroimmunoassay (TRFIA) is the rare earth ion labeled antigen or antibody, nucleic acid probes and cells, characterized by a new type of non-radioactive immune markers. TRFIA overcomes several shortcomings like the instability of the ELISA enzyme markers; O.D values only in the low concentration range of enzyme activity was a linear relationship; Hook; the enzyme activity easy to lost inactivation; low sensitivity. TRFIA achieves a high signal to noise ratio, greatly exceeds the sensitivity of radioisotope. It also has a simple preparation of markers, and long storage time, no radioactive contamination detection repeatability, short operating procedures, a wide range of standard curve from samples of natural fluorescence interference and wide range of applications, etc. TRFIA has become a new milestone after the development of the radioimmunoassay, it has become a biomedical research and clinical ultramicro amounts biochemical tests, one of the most promising development in analytical tools, will be expected to take the place of NIH method for the detection of rabies vaccine in future. Methods:Double antibody sandwich method developed rabies virus N protein quantitative detection reagents to assess performance indicators. To prepare rabies virus G protein monoclonal antibodies by hybridoma fusion.1. Development and application of human rabies virus nucleoprotein quantitative detection reagents.1.1Preparation of rabies virus nucleoprotein antigen:The complete length of nucleoprotein (N) gene of the CTN rabies virus vaccine strain was amplified by PCR using a pair of specific primers designed according to the relevant sequences from GenBank, then it was cloned into plasmid p ET32a (+) to obtain the prokaryotically expressed plasmid p ET32a/N. The target gene was then expressed in the Escherichia coli BL21(DE3) cells by inducted with IPTG and the expression was optimized with a proper induction time of4hours. The target protein was identified by SDS-PAGE and Western-blot.1.2Preparation of human rabies virus anti-N protein monoclonal antibodies: Bal/C mice were immunized with rabies virus protein. When the titer reach1:64000, prepared monoclonal antibodies by hybridoma fusion method. And then screened antibodies with NP, to sieve out of the14anti-NP monoclonal antibodies.1.3Purification of human rabies virus anti-NP monoclonal antibody:Purified with Protein G Sepharose4Fast flow affinity chromatography column, and the BCA kit for measuring antibody concentration.1.4Europium labeled antibody and purification:Anti-N protein antibody were0.5mg of adding50KD protein ultrafiltration spin columns,9000r/min centrifugation8min. The then labeling buffer (50mmol/L, Na2CO3, pH9.0) is repeatedly washed six times.200μl of labeled antibody and50μg europium labeling reagent thoroughly mixed overnight at25℃with shaking. Mark the completion of the protein ultrafiltration spin columns,9000r/min centrifugation8min. Upside down after repeated elution then the eluent (including0.9%NaCl50mmol/L Tris-HCl) Discard Finally, add500μl of eluent for2min,9000r/min centrifugation5min, collect the filtrate.1.5Monoclonal antibodies screening:First with the rabies virus stock as the standard double-antibody sandwich,14anti-NP monoclonal antibody-coated, with another13Eu standard, standard curve, sieve out the pairing on singleresistant, then homemade NP and corporate standards as the standard from the standard curve, the final screen pairing on monoclonal antibodies.1.6Kit performance evaluation:Accuracy test (dilution recoveries), the linear range of detection, sensitivity (minimum detectable amount), precision testing, stability testing, home-made reagents and other reagents comparison.1.6.1Accuracy test (dilution recoveries):Sample1, Sample2, Sample3press1:200,1:400,1:800and1:1600-fold dilution of each test for3wells, repeated measurements of3times, n=3×3, and calculating the measured value, the true value, standard deviation and recovery.1.6.2The linear range of detection:Reference standard antigen was diluted to different concentrations were measured.3wells each concentration of test values for the test three times, n=3×3, calculating the measured value of the test, the true value of their ratio, and the analysis of linear regression and calculate test the correlation coefficient r.1.6.3Sensitivity (minimum detectable amount):Standard Diluent is used as a blank zero (A) as a sample measurement,8wells each test set, repeated three times, n=8×3, calculate the mean and standard deviation.1.6.4Precision testing:Analysis precision:the enterprise standard AF points each doing10wells, calculate the mean, standard deviation, and CV values; interassay precision:enterprise standard AF point by three different techniciansto operate, each doing10wells, calculate the mean, standard deviation, and CV values.1.6.5Stability test:the homemade kits were placed in4℃refrigerator for six months, and37℃7days, the physical appearance of the kit under various conditions, the dose-response curve linearity, accuracy, precision, limit of detection,specificity and other indicators were measured.1.6.6Comparison with the measured value of to with Wuhan Institute rabies virus N protein ELISA kit:homemade kit with Wuhan as ELISA rabies virus detection kit for sample measurement, and calculate the correlation coefficient.2. Preparation of rabies virus G protein monoclonal antibody:Bal/C mice by rabies virus vaccine titer reaches1:64000hybridoma fusion method to prepare monoclonal antibodies with rabies virus stock and GP screened positive strains, NP, then the laboratory homemade human albumin antibody anti sieve, sieve out of the48anti-GP monoclonal antibodies.Results:The success of the rabies virus N protein gene was cloned into the p ET32a (+) prokaryotic expression plasmid, identified by PCR, restriction enzyme digestion and sequencing, successfully built p ET32a/N prokaryotic expression plasmid. The recombinant plasmid p ET32a/N expressed in E. coli of a fusion protein of the rabies virus N protein, a molecular weight of about70kDa, found with the theoretical values, the expression product is mainly in the form of inclusion body. The recombinant proteins after the inclusion body was washed Ni affinity chromatography and after purification, purity more than90%. Expression product of Western-blot analysis with anti-His tag antibody found The specific bands70kDa at the show that the protein is indeed to express the fusion protein. Immunization of mice with purified rabies virus, the three immune aftereffect price reaches1:64000, cell fusion to prepare the14anti-NP monoclonal antibody, ELISA results show:the self-made monoclonal antibodies capable of reacting specifically with the natural protein described the preparation of monoclonal antibodies are specific mAb. F023get to use the method of time-resolved screening and F022paired antibodies on antibody preparation of rabies virus N protein kit. NP kit accuracy, the linear range of5to2500mEU/ml sensitivity of1.018mEU/ml, good precision, within-run precision of2.7%to9.3%between the precision of3.5%to12.2%. Stability tests showed that the reagent is stable for six months at4℃,37℃stable for7days. Comparison of measured values with the Wuhan Institute of rabies virus N protein ELISA kit, the kit detection results with Wuhan N protein kit tends to be highly consistent. Bal/C mice by rabies virus vaccine titer reaches1:64000, using the method of preparation of monoclonal antibody hybridoma fusion, screening positive dope and GP with rabies virus strain, and then laboratory made the NP, human albumin of antibodies anti-sieve, sieve out of the48anti-GP monoclonal antibodies, ELISA results show:the self-made monoclonal antibodies with the natural protein can react specifically described the preparation of monoclonal antibodies are specific mAbs.Conclusion:These results suggest that this study successfully build p ET32a/N prokaryotic expression plasmid expression, having a large number of immune activity of the recombinant proteins in Escherichia coli secretion; screening, identification obtained14cell lines antibody-producing hybridomas of rabies virus anti-NP monoclonal and screened to obtain a pair antibodies, the indicators developed using this antibody with the rabies virus N protein quantitative detection reagents (accuracy, sensitivity, precision, stability, etc.) have reached clinical detection reagent requirements, is expected to replace the sample testing of domestic and abroad. Successful rabies virus anti-GP monoclonal antibodies48provide the raw materials for the rabies virus G protein immunological detection reagents.