| Tick-borne encephalitis virus is a kind of virulent virus of Flavivirus, mainly transmitted by ticks, human body infected with TBEV could cause severe clinical symptoms and threatens human health and social stability. There are three closely related subtypes, European, Siberian, and Far Eastern respectively, and the main subtype of TBEV in our country is Far Eastern, the fatality rates is high at approximately 20%, therefore the clinical diagnosis of tick borne encephalitis virus infection is important to the control and treatment of the disease. The genome of TB E virus is a positive-stranded RNA, encoding three structural proteins and seven nonstructural protein. The envelope glycoprotein(E) is the first antigen for TBEV- infected detection, which is composed of 496 amino acids and contains 90% antigen neutralization epitope of natural virus. Up to now, the mainl used antigen for TBEV- infected serum detection is whole virus derived by virus culture and the envelope glycoprotein expressed in vitro.In this study, we expressed the extracellular region of envelope glycoprotein and evaluated its effect in TBEV serum detection. Then used VE3 as detecting antigen, a new TBEV- infected serum detection technology- Luciferase immunoprecipitation system(LIPS) was established and optimized. The gene of luciferase and antigen were co-expressed in eukaryocyte to produce the recombinant antigen, and the antibody levels were measured in sample by detecting the luciferase activity in a mixture of the detection antigens-antibody-agarose gel beads. The advantages of this method are high detection sensitivity and easy to operate.The main contents of this study are listed as following: 1. Optimization and expression of the envelope extracellular region in eukarya system, and the evaluation of the detection effect in serological assayFirst, we analyzed the antigenic domain of TBEV envelope glycoprotein(E protein), and chose the extracel ular region of envelope glycoprotein(EC) from TBEV-MDJ01 as antigen for the LIPS detection of TBEV infected serum. Optimized the gene of EC by www.jcat.de to obtain the optimization sequence of the extracel ular region of envelope glycoprotein(OPT-EC), and the gene sequence of OPT-EC was obtained by gene synthesis. The gene of antigen and luciferase were inserted into the vector to construct the recombinant plasmid, cel s were transfected by recombinant plasmid to collect the recombinant antigens of Rluc-EC and Rluc-OPT-EC. The luciferase activity could represent the relative expression level of recombinant antigens. Compared the expression level of Rluc-EC and Rluc-OPT-EC, meanwhile utilized the recombinant antigen as detection antigens in the LIPS assay of TBEV infected serum and evaluated the sensitivity and specificity of antigens.The luciferase activity test of Rluc- EC and Rluc-OPT-EC showed that the LU of Rluc-OPT-EC was about 12-fold higher than Rluc- EC demonstrated that the expression level of Rluc-OPT-EC was higher than Rluc-EC. Used the recombinant antigens Rluc-OPT-EC in the detection of TBEV- infected serum by LIPS assay, the positive detection rate of OPT-EC was upper than 86%, thus in the detection of the JEV infection serum, YFV infection serum, AIV infection serum and normal human serum, the detection specificity was higher than 90%, but there were cross reaction in the detection of the WNV infection serum and DENV infection serum.The results showed that the eukaryotic codon optimization can effectively improve the expression level of heterologous protein in eukaryotic expression system, and the optimized extracellular region of envelope glycoprotein as TBEV- infected detecting antigen can effectively distinguish the JEV, YFV infection, but could not distinguish the infections in WVN and DV. 2. Establishment and optimization of a new type LIPS assay1) Construction of COS7 cell lines which could stably express TBEV detecting antigensThe detection antigen of LIPS assay were prepared by transient transfection, which is a long-term and reagent-consuming experiment, in order to further simplify the preparation method of eukaryotic antigen, used the lentiviral vector system to construct a cell line which could stably express TBEV detecting antigens. The gene of Rluc-VE3 and Rluc-OPT-EC were inserted into the lentiviral vector to construct the recombinant plasmid, and the recombinant lentiviral plasmid and the packaging plasmid(pska and pMD?2D) were co-transfected into 293 Tcells to package the lentivirus. Lentiviral particles can infect the eukaryotic cells(COS7) to integrate the virus genome into the cell genome. The cell line was secreted by RFP and Puromycin, and obtained new cell lines which could stably express the recombinant antigens Rluc-VE3 and Rluc-OPT-EC. Test the luciferase activity of the antigen produced by cell lines, and the antigen were also used in the detection of TBEV- infected serum to evaluate the detection effect.The lentiviral vector contains the gene of RFP and Puromycin, therefore the stable cells with red fluorescence protein and Puromycin resistance demonstrated that the lentivirus genome was integrated into the cell genome. For further verification, we test the luciferase activity and antigenicity of the antigens produced by cell lines. The luciferase activity test showed that the LU(light units) of Rluc-VE3 was 5.14×106LU, and the LU(light units) of Rluc-OPT-EC was 4.29×105LU, so the expression level of Rluc-VE3 was higher than Rluc-OPT-EC, the large weight of Rluc-OPT-EC probably affect the expression level. The Rluc-VE3 was used in the TBEV-infected serum detection to determine the antigenic of the recombinant antigens. In the LIPS assay of 19 TBEV- infected serum, 13 serum were positive, the positive detection rate was 76.47%(**p<0.01). However, in the early stage of laboratory work, the positive detection rate of the transfected antigen Rluc-VE3 was 79.2%, the positive detection rate of the transfected antigen and the stable antigen was similar, which implied that the recombinant antigen Rluc-VE3 produced by stable cell lines could be used as detection antigens in the LIPS assay of TBEV-infected serum.In present study, we constructed COS7 cell lines which could stably express TBEV detecting antigens, simplified the antigen preparation method, reduced the experimental cost and operation time, sustained and stable for TBEV infection was detected in the sera provides diagnostic antigen, and provided stable diagnostic antigen for the LIPS detection of TBEV-infected serum.2) Nanoluciferase as a protein fusion tag in LIPS assayIn the experiment operation of the LIPS assay, we found that the thermal stability of Renilla luciferase was low, the half- life of Rnilla luciferase is 10 mins in 37°C, so the incubated temperature of antigen and antibody was RT(room temperature). However, the best binding condition of the antigen and antibody is 37 °C, this temperature could shorten the binding time and improve the detection sensitivity. To break the limit of the environmental temperature of Renilla luciferase, we chose Nano luciferase to replace Renilla luciferase as protein fusion tag in LIPS assay. Nano luciferase is a new genetically engineered luminescent reporter. Nano luciferase has the advantages of small molecular weight, strong fluorescence signal. In addition, Nano luciferase fused to an N-terminal secretion signal, therefore, the expression of Nano luciferase is extracellular secretion, so the cells could secrete the recombinant antigens out of the cells and the antigen is in the supernatant. But the expression of Renilla luciferase is intracellular secretion, so the antigen is collec ted by lysing cells, the antigens in supernatant contains less miscellaneous proteins than the cell lysis solution, which could improves the detection sensitivity. So insert the gene of Nano luciferase and the gene of antigens into vectors to produce the recombinant antigen Nanoluc-VE3, and test the luciferase activity, thermal stability and the antigenicity of Nanoluc-VE3.We produced the recombinant antigen Nanoluc-VE3 by eukaryotic cells, and compared the thermal stability of Rluc-VE3 and Nanoluc-VE3. The result demonstrated that the luciferase activity of Nanoluc-VE3 is stable both at 37 °C and RT. The test of the luciferase activity showed that the LU of Nanoluc-VE3 was 7.79×109LU, and the LU of Rluc-VE3 was 6.71×106LU, which demonstrated that the the fluorescence signal of Nanluc-VE3 was significantly higher than Rluc-VE3.Used the Nanoluc-VE3 as detection antigen in LIPS assay of 30 TBEV- infected serums, there were 28 serums were positive, the result demonstrated that Nanoluc-VE3 has the antigenicity of VE3 and Nanoluc-VE3 could be utilized in the LIPS assay of TBEV- infected serum. Based on that, we constructed a new cell line which could stably express recombinant antigen Nanoluc-VE3. The stably expressed Nanoluc-VE3 was also used in the LIPS assay, the positive detection rate of 30 TBEV- infected serums was 93.33%. The positive detection rate of Nanoluc-VE3 is much higher than Rluc-VE3(76.47%), which demonstrated that the sensitivity of Nanoluc-VE3 is higher than Rluc-VE3. The detection specificity of JEV infection serum and DV infection serum was 100%. As a consequence, Nano luciferase could be a protein fusion tag in LIPS assay, this part provides a new type of fluorescent protein for LIPS assay.The present study evaluated the effect of extracellular region of E protein as diagnostic antigen in LIPS assay for TBEV- infected serum detection, and provided important experimental basis for antigen screening of TBEV serological detection. For optimization of LIPS technology, a more stable and sensitive luciferase-Nano luciferase, was used as a marker in LIPS assay. Furthermore, we constructed cell lines which could stably express foreign proteins. LIPS detection method is simple r and less time-consuming. This study provides a new antigen for diagnosis o f clinical sera infected with TBEV, which has an important role for the rapid detection of TBEV-infected serum. |
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