Expression And Application Of Hantavirus Recombinant Nuclear Protein In Hemorrhagic Fever With Renal Syndrome

Objective:The recombinant nucleoprotein of Hantavirus was constructed and induced in Escherichia coli（E.coli）BL21（DE3）,the prokaryotic expression system,and purified.The purified protein was used in the preparation of colloidal gold immunochromatographic strips for detecting Ig M and Ig G antibodies of hemorrhagic fever with renal syndrome to provide technical reserve for the early and rapid diagnosis of hemorrhagic fever with renal syndrome.Method:1.The whole S gene fragment of Hantavirus Jiangxi xinjian Rn09-2011 strain（XJ9 for short）stored in our laboratory was amplified by RT-PCR,and TA clone vector and p ET30a-XJ9S expression vector were constructed,which were identified by PCR and enzyme digestion and sequenced.The antigenicity of the recombinant nuclear protein was determined by ELISA and Western blot.2.The purified recombinant nuclear protein was used to prepare colloidal gold labeled antigen,optimize the p H of the gold labeled antigen probe,the amount of antigen labeled and the amount of probe dots,optimize the chromatographic time of the strip,the p H and concentration of the coated antibody,test the sensitivity,specificity,stability and accelerated preservation time of the strip,and apply it to the detection of serum samples and the preliminary test of urine samples.Results:1.The positive TA clone and p ET30a-XJ9S expressed Escherichia coli were successfully constructed,and the induced expression of recombinant nuclear protein p ET30a-XJ9S was significantly affected by temperature,while the IPTG concentration and induction time had little effect;The recombinant nuclear protein was mainly soluble supernatant expression protein,and the expression level of p ET30a-XJ9S was the highest when the recombinant nuclear protein was induced by0.2 mmol/L IPTG at 20℃for 14 h.The purified recombinant nuclear protein showed good antigenicity.2.Optimized results of immunochromatographic conditions of colloidal gold:（1）The appropriate p H of gold-labeled antigen probe was 6,the required amount of antigen was 8μg,and the volume of gold-labeled antigen required for test paper was5μL;（2）The optimum chromatographic time was 15 min,the optimal p H of the buffer solution used for coating antibodies on quality control line C and detection lines T₁and T₂was 7,and the optimal coating concentration of antibodies on quality control line C,detection lines T₁and T₂was 0.3 mg/m L,0.1 mg/m L and 0.1 mg/m L,respectively.（3）The sensitivity of the colloidal gold immunochromatographic strip is high.For one HFRS Ig M and Ig G strongly positive serum,the maximum dilution of the colloidal gold immunochromatographic strip for positive color development is500 times,and the maximum dilution of the commercial strip for positive color development is 400 times;For one sample of HFRS Ig M and Ig G weakly positive serum,the maximum dilution times of positive color development of colloidal gold immunochromatography strip was 150 times,and the maximum dilution times of positive color development of commercial strip was 100 times.There was no color on the T line in the serum of patients infected with 8 other viruses and healthy people.The results of the same batch and different batch of colloidal gold immunochromatographic test strips were consistent for the same sample.Strong stability,45℃storage period is 28 days;（4）The Ig M and Ig G results of 50 suspected hemorrhagic fever with renal syndrome were 100%consistent with those of commercial strips,but the Ig M color of 9 serum samples and the Ig G color of 1 serum sample detected by commercial strips were weaker than that of prepared colloidal gold immunochromatographic strips.The Ig M positive rate and Ig G positive rate were 30%and 70%respectively in 10 urine samples from suspected patients with hemorrhagic fever with renal syndrome.Conclusion:In this study,the prokaryotic expression system of Hantavirus nucleoprotein strain xinjian Rn09-2011 was constructed,and purified Hantavirus recombinant nucleoprotein was obtained.Colloidal gold immunochromatographic strips for detecting Ig M and Ig G antibodies of hemorrhagic fever with renal syndrome were successfully prepared using the recombinant nucleoprotein,which was suitable for the detection of serum samples.The presence of Ig M and Ig G antibodies in urine was confirmed by the tentative detection of urine samples from suspected patients with hemorrhagic fever with renal syndrome,which provided a practical basis for the later development of non-invasive urine test strips and a technical reserve for the early and rapid diagnosis of hemorrhagic fever with renal syndrome.