The Application Research Of Hantavirus S Segment Coding Producetion Used In Epidemic Haemorrhage Fever Early Diagnosis

Hemorrhagic fever with renal syndrom(HFRS),which is caused by hantavirus,is an acute infectious disease characterized by fever,shock,hemorrhage and kidney dysfunction.Since Dr.Li have separated the Hantavirus 76-118 strain from epidemic area,several academician have separated different kind of Hantavirus on after the other.Now,Hantaan(HTN),Seoul(SEO),Puumala(PUU),Sin Nombre(SNN), Dobrave(DOB) have been identified in the world.HTN and SEO are the main rarity in China.About 40000-70000 cases of HFRS are reported yearly in China,where is a severe area of HFRS in the world.And now,there have been no specific drugs for treating HFRS yet.Hantaviruses are tripartite negative-standed RNA in the Bunyaviride family.The viral RNA segments,L,M,and S,encode an RNA dependent RNA polymerase,two envelope glycoproteins(G1 and G2) and nucleoprotein(NP).Respectively,the nucleoprotein have high homologous in all kinds of Hantavirus,and can induce the body fluid immunity responsion and cell immunity responsion.It is the best antigen in the early diagnosis.The purpose of our research is to construct Hantavirus 76-118 S gene sequence, which is epidemic in China,then we use it in Real-time PCR and CGIDA to the early diagnosis for acute patients,and compared with the methods of ELISA,IFA to ensure the important function in diagnosis and cure Hemorrhagic fever with renal syndrom.Partâ… :Construction of Referance Standard Plasmids of Real-time Fluorescence Quantitative PCR for Haantan Virus S segment and the Reseach of the Application in Early DiagnosisTo establish a sensitive and specific method of assay for quantitative detection of Hantavirus RNA in serun of patients with hemorrhagic fever with renal syndrom (HFRS),and to explore the function of nucleoprotein,we find the method of preparation for hantavirus S segment standard plasmids of real-time PCR and used to detect hantavirus RNA.Firstly,analyse S gene rearrangement sequence of 76-118 stains.Obtain a couple of primer and fluorescent probe from the primer expression software.The standard recombinant plasmids was gained from the positive plasmids. RT-PCR,agarose gel electrophoresis,gel extraction,T-A clone,sequance,plasmids nimiprep,quantify.RNA was extracted from the acute patient,RT-PCR,The PCR product was detect by real-time PCR with the standard plasmids all together.And at the same time detcet the acute patients antibody by IFA.The research results indicate: we get steady stanard plasmids with this method,and take successfully quality control in early detection with the standard plasmids;With the method of real-time PCR,the acute patients can be detected quickly and precisely.The fluorescence levels were increased incisively after real-time quantitative PCR amplification,which indicated that S segment was cloned successfully.This method is suitable for hantavirus RNA detect and it is a good way to detect hantavirus in the early time of patient,and NP is the idea antibody of HFRS early diagnosis.Partâ…¡:Expression and Purification of the Recombinant Core Area of Hantavirus S Segment Nucleocapsid Proteins.To prepare the ideal,stable and unique recombined nucleocapsid Protein of Hantavirus,we cloned three core segments area of S gene of Hantavirus HTN type 76-118 strain in prokaryotic expression plasmids.Hantvirus S gene ORF of HTN type 76-118 strain was amplified by PCR and use the primer offered by NCBI from the primer expression software.We extract the totally RNA of HTN hantavirus from Vero-E6 cell infected with 76-118 strain,after PCR,enzymatic cut,connect and translate,the antigen of rNP was prepared by prokaryotic expression and purification of the core sequence of the small(S) genome segment of Hantan stain HTN with the expression vector pGEX-4T-1-S1,pGEX-4T-1-S2,pGEX-4T-1-S3.The sequence results indicate:The sequnce of recombination is totally correct.After constructed the prokaryotic expression vector,we express the protein in E.coli and analyzed by using SDS-PAGE and Western-blot.The results of analysis showed that there is only one special antigen about 38KD with 167aa have sucessfully expessed and induced by IPTG.This recombination protein existed in the E.coli about 12KD,and the GST is about 26KD,and the GST have no infection on the function and activity of recombinated protein.We have approved the successfully expression of prokaryotic vector and got the high purification antibody.Recombinant NP is effective and valuable antigen used in diagnosis of HFRS.Partâ…¢:The Application of Recombined Nucleocapsid Proteins used in Epidemic Haemorrhage Fever Early DiagnosisEpidemic Haemorrage Fever is a kind of urgent infectious disease caused by Hantavirus.Since the sucessful separation of HV,many specific detect methods have been quickly established.The ELISA method have benn used in detecting IgM and IgG antibody of virus;the IFA method used in detecting IgM and IgG antibody in pateints' blood.These two methods are the main means in laboratory today. During the acute infection period,4 times of IgG increased of patients' double blood is the standard of diagnosis of HFRS.The method of CGIDA have also used to detect many infectious disease,it has the merit of easyly,quickly and specially.In order to construct a method used in early diagnosis and supervision and especially adapt to the basic units,we make use of recombinant protein of NP in CGIDA and establish the detection method of CGIDA.We prepared CGIDA detection reagent,and establish the same system of CGIDA compared with the HFRS acute patiens' blood by using of recombination NP and inartificial NP.The method was also compared th those of specific antibodies by IFA for IgG and ELISA of IgM.The results shows:The coincidence rate of recombinant protein and natural protein was 89.7%for detecting IgM and 92.3%for IgG.300 sera from pateints with HFRS and 100 sera from patients of other diseases were tested by CGIDA,IFA and ELISA.When compared with ELISA,the specificity of CGIDA for detection of IgM was 100%,and the sensitivity was 75.0%.Compared with IFA, the specificity of CGIDA for detection of IgM was 100%,and the sensitivity was 83.1%.So we get the conclusion that recombiant NP is effective and valuable antigen used in CGIDA for diagnosis of HFRS.