| Background:Rabies virus(Rabies virus,RABV)causes rabies to occur in humans and animals,causing 100%of the death of the host,and a serious threat to public health safety.The internal core composition of rabies virus particles is the combination of nucleoprotein and viral RNA to form a nucleocapsid complex.In addition to being an important part of rabies virus particles,the nucleoprotein is also responsible for regulating virus replication and activating host immunity.It can also be used for in vitro testing,virus typing,etc.Obtaining high-purity nucleoprotein is the basis for basic scientific research and applied research.At present,scholars have used eukaryotic systems to recombinantly express nucleoprotein,but the cost is high,the operation is complicated,and it is difficult to obtain a large amount of nucleoprotein.Other scholars use the prokaryotic system expresses nucleoproteins,mostly in the form of inclusion bodies.Some people try to use fusion tags or truncated expression of some of the sequences,but it is difficult to obtain nucleoproteins with biological activity.In this experiment,the nucleoprotein coding sequence was optimized on the basis of the CTN-1 strain,and different expression conditions were used to promote the soluble expression of nucleoprotein,in order to obtain high-yield,high-purity nucleoprotein with biological activity,and carry out initial application.Methods:1.Extract the total RNA of rabies virus CTN-1 strain,amplify the full-length N gene of rabies virus CTN-1 strain by RT-PCR,add Nde I and Xho I sites at both ends of the gene to construct a recombinant plasmid pET-43.1 a-NP.The expression was induced in Escherichia coli BL21(DE3)strain,and the expression results were analyzed using SDS-PAGE and Image Lab software.2.The recombinant plasmid pET-43.1 a-NP was not expressed in E.coli.The N gene sequence was optimized.By replacing rare codons,adjusting the GC content,optimizing the secondary structure of mRNA,artificially synthesized the gene to construct pET-43.1 a-CTN-NP(Opti)expression plasmid.By changing the induction conditions,optimizing the induction time and increasing the expression yield of nucleoprotein.According to the optimal expression conditions,a large number of Escherichia coli was induced,lysed and sonicated the induced bacterial solution,and the supernatant and precipitate were collected after centrifugation.The precipitate was dissolved with guanidine hydrochloride,and immobilized N2+chelation and chromatographic purification were used.By changing the buffer formulation,the recombinant nucleoprotein is renatured at low temperature.3.In order to obtain the soluble nucleoprotein of RABV CTN-1 strain expressed by E.coli,the induction temperature,IPTG concentration and induction time were explored.Purified by Ni2+chelating chromatography column,verified the band position by SDS-PAGE and verified the biological activity by Western Blot.The soluble nucleoprotein,the refolded nucleoprotein,the negative control,and the positive control were injected into the mice,and the mouse serum was collected,and the immunogenicity of the recombinant nucleoprotein was detected with a commercially available rabies nucleoprotein antibody detection kit.4.Based on the reactogenic and immunogenic nucleoprotein expressed in this study,the optimization of coating concentration,coating temperature,coating time,blocking solution,blocking conditions and second antibody concentration,an indirect ELISA method that can detect rabies virus antibodies was established,and the practicability and reproducibility of the method were preliminarily verified with known samples.Results:1.The recombinant expression plasmid of nucleoprotein of rabies virus CTN-1 strain was constructed and induced in Escherichia coli.SDS-PAGE showed that there was no significant difference in the expression of bacterial fluid after induction and before induction.Therefore,further optimization of gene sequence was needed to improve the expression yield.2.The N gene of the rabies virus CTN-1 strain was optimized.Compared with the original sequence,the GC content of the optimized sequence was increased to 51.5%,all 61 rare codons were replaced,and the codon adaptation index(CAI)value of the N gene was increased from 0.19 To 0.95.After software analysis,the mRNA secondary structure of the optimized sequence was more stable and the hairpin structure was reduced.3.The optimized N gene was induced to express in Escherichia coli,and obvious protein bands appeared at about 50 kD,and the expression yield was significantly increased.In order to further improve the nucleoprotein expression yield,the induction time and Escherichia coli OD600 were optimized.The optimal expression condition was found that when the OD600 was 0.5 and the induction time was 5 h,and the yield could reach 32.3%.After large amount of induced expression of Escherichia coli,the nucleoprotein was expressed in the inclusion body.After purification,the protein purity was 95.8%.By optimizing induction temperature,IPTG concentration and induction time,and after verification,it was found that the soluble nucleoprotein could be obtained when E.coli was induced overnight at 16?.4.The nucleoprotein showed obvious bands at about 50 kD after Western Blot detection,indicating that the nucleoprotein can be bound by specific anti-nucleoprotein antibodies and it had good reactivity.After immunizing mice with recombinant nucleoprotein,serum was collected.The antibody titer reached 1:320,showing good immunogenicity.5.The verification results of known serum samples suggest that the indirect ELISA qualitative test established in this research have a negative and positive coincidence rate of 100%,and the coefficient of variation(CV)of intra assay and inter assay were both less than 10%.Conclusion:In this study,the recombinant expression plasmid pET-43.1a-CTN-NP(opti)was constructed by codon optimization of the nucleoprotein of CTN-1 strain of rabies virus under the condition that the original sequence expression yield was low.The soluble expression of nucleoprotein in Escherichia coli expression system was realized by lowering the induction temperature and prolonging the induction time.Western Blot and animal test results show that the nucleoprotein we expressed has good reactogenicity and immunogenicity.On this basis,this study also initially established an indirect ELISA detection method that can detect rabies virus antibodies. |
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