The Preparation Of Chikungunya Virus-like Particles And The Preliminary Establishment Of A Specific IgM Detection Method

【Background】Chikungunya virus（CHIKV）,an arbovirus transmitted by the mosquito vector,is a member of the alphaviruses within the Togaviridae family.The genome of CHIKV is a single strand,positive RNA,encoding 4 Non-structural proteins（NSPs）and five structural proteins: C,E3,E2,6K,E1 protein.The E2 protein is the major antigen for neutralizing antibodies.Infection of CHIKV through mosquito（Aedes aegypti and Aedes albopictus）causes Chikungunya fever（CHIKF）characterized by fever,rash and severe and sometimes prolonged polyarthritis.Initially,CHIKV was mainly popular in Africa and Southeast Asia.Since 2004,CHIKV has caused millions of cases in the Indian Ocean region and occured in more areas,including Europe,the Middle East,and the Pacific region.In October 2013,CHIKV appeared in the Western hemisphere for the first time,and spread to 22 countries in Central and South America in just nine months,resulting in more than one million CHIKF cases.CHIKF is a self-limited disease which has low lethality,but resulting in severe damages to the joints and enervating patients.Considering the wide distribution of the mosquito vectors,CHIKV infection has become one of the most important global public health problems.Recently,there was no outbreak of CHIKV in China but sporadic imported cases.However,our country is still under the risk of CHIKF epidemic because of the wide distribution of Aedes albopictus and Aedes aegypti.Thus,it is still an urgent need for the diagnosis,prevention and treatment of CHIKV infection research.Currently,the virus-specific IgM is a major marker for early infection due to the short viremia.The most accurate and commonly used CHIKV detection method is the IgM antibody capture enzyme-linked immunosorbent assay（Mac-ELISA）,which avoids being affected by the IgG、rheumatoid factor（RF）and anti-nuclear antibody（ANA）in the samples.There are several commercialization kits making use of the Mac-ELISA to detect CHIKV infection abroad,but most of them employ inactivated virus as the antigen,resulting in the uneven quality and certain bio-safety risk.Whereas in China,there is still no available commercialized kit for CHIKV IgM detection.Our present research took advantage of the Baculovirus expression vector system（BEVS）to express the CHIKV virus-like particles（VLPs）,serving as the antigen of Mac-ELISA.This detection method lays the foundation of the CHIKV infection diagnostic kit in future.【Methods】The primers were designed to amplify the structural protein gene and E2 protein gene of CHIKV（strain BNI-CHIKV₈99）,then the two gene fragments were cloned into pFastBac?-Dual vector and pFastBac?-His A vector to construct recombinant transfer vectors（pFastBac?-Dual-CHIKV and pFastBac?-HisA-E2）respectively.The two recombinant transfer vectors were confirmed by restriction enzyme digestion and sequencing,then transformed into DH10 Bac competent cells to isolate two recombinant bacmids（r Bacmid-CHIKV and rBacmid-E2）respectively.The two recombinant bacimds were confirmed by PCR amplification with M13 universal primers and specific primers,then transfected into Spodoptera frugiperda9（Sf9）insect cells to produce two recombinant baculovirus（rBV-CHIKV and rBV-E2）.Immunofluorescence assay（IFA）was performed to identify the two recombinant baculovirus.The Sf9 cells were transfected with the rBV-CHIKV.After the cytopathic effect（CPE）emerging,the expressed VLPs in the cells were observed by the transmission electron microscopy（TEM）.The intracellular CHIKV VLPs were collected by repeated freezing and thawing cells extensively,then VLPs were purified by sucrose density gradient centrifugation.Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）and Western blotting（WB）were used to identify the CHIKV VLP.The Sf9 cells were transfected with the rBV-E2.After the CPE emerging,the supernatant was collected and purified by Nickel beads adsorption method to get the E2 protein.SDS-PAGE and WB were used to identify the E2 protein.The purified E2 protein was injected to immune BALB/c mice for three times.The spleen cells of the immuned mice and Sp2/0 cells were fused with PEG1500 to prepare the hybridoma cells.Enzyme-linked immunosorbent assay（ELISA）was used to screen positive hybridoma cells.The positive hybridoma cells screened by ELISA were cloned by limiting dilution method,then the confirmed hybridoma cells were pick out for expanded culture.The rBV-CHIKV-infected Sf9 cell antigen was used to identify the activity of monoclonal antibody（mAb）,and the cells with better activity were selected for further experiments.Finally,hybridoma cells were injected into BALB/c mice intraperitoneally for antibody preparation.Mice ascites were collected and purified by octanoic acid-saturated ammonium sulfate method to obtain purified anti-E2 mAb.SDS-PAGE was used to determine the purity of the mAb.The antigen of Hantaan virus（HTNV）,Dengue virus（DENV）,Japanese encephalitis virus（JEV）,Herpes simplex virus Virus I（HSV-1）and Herpes simplex virus Ⅱ（HSV-2）were used to identify the specificity of the mAb.The reactivity with CHIKV VLP was detected by WB and indirect ELISA.Based on above works,we initially established the CHIKV VLP-based IgM capture ELISA（Mac-ELISA）with simple,fast,safe,sensitive and specific characteristics.This method used a solid carrier coated with anti-human IgM antibody（μ chain specific antibody）which acted as the "capture antibody" to capture the IgM in the sample.Then CHIKV VLP was added to bind with the corresponding IgM antibody.Finally,the mAb was used to react with the VLP.The absorbance was measured at 450 nm by adding the enzyme-labeled secondary antibody and TMB.The same positive serum was tested by gradient diluted VLP and antibody.The optimal concentration of VLP and antibody was determined according to the experimental data.The serums of CHIKF patients、HFRS patients、Japanese encephalitis patients and healthy person were tested by Mac-ELISA to determine the specificity of the method.Two high-OD and low-OD serums were serially tested four times to determine the repeatability of the method.Finally,the serums of positive patients and healthy person were tested by the VLP-based Mac-ELISA and the commercial testing kit.The results of the two groups were compared and analyzed.【Results】 1.Preparation,purification and identification of CHIKV VLP and E2 proteinsAfter the structural protein gene and E2 protein gene were respectively cloned into pFastBac?-Dual vector and pFastBac?-HisA vector,we used EcoR Ⅰ and Not Ⅰ enzyme to authenticate them.The results of Agarose gel electrophoresis suggested that we has constructed the two recombinant transfer vectors.The sequencing results further confirmed the construction.The PCR results of r Bacmids suggested that we has constructed the two recombinant bacmids.The results of IFA suggested that we has produced the two recombinant baculovirus.After Sf9 cells were infected with the rBV-CHIKV,we observed that the particles ranging from 60 nm to 90 nm in diameter distributed in cells under electron microscope.The VLPs collected from lysis cells were concentrated by sucrose density gradient and measured at a concentration of 1 mg/ml.SDS-PAGE and WB results showed specific bands corresponding to the size of target protein.After Sf9 cells were infected with the rBV-E2,the supernatant was purified by Nickel beads and detected by SDS-PAGE and WB.The specific bands consistent with the size of target protein could be detected in the supernatant.2.Preparation and identification of anti-CHIKV E2 mAbThe purified E2 protein was injected to immune BALB/c mice,followed by cell fusion,selective culture,positive hybridoma cells identification and 3 subclones.Six hybridoma cell lines secreting anti-E2 mAb were identified and named as 3B1,2B8,4A4,3G9,2H4 and 2D2.The IFA results of the analysis of activity showed that the antibody activity of 2H4 cell line was better than others.The 2H4 hybridoma cells were collected to produce ascites.The purified antibody was measured at a protein concentration of 10 mg/ml.SDS-PAGE showed that the purified antibody had two specific bands at 55 KD and 25 KD,and other bands were less observed.The results confirmed that a mAb of high purity was obtained.IFA results showed that none of the HTNV,DENV,JEV,HSV-1,HSV-2-infected cells showed fluorescence,indicating that the mAb had a high specificity.WB result showed that the purified 2H4 mAb group had a significant band at about 47 KD,which was consistent with the size of E2 protein.Whereas the negative control group had no band,indicating that 2H4 mAb could specifically recognize CHIKV VLP.Indirect ELISA results showed that the titer of purified 2H4 antibody was 1: 100,000.3.Preliminary establishment of a specific IgM detection method based on VLPBased on above work,we initially established the CHIKV VLP-based IgM capture ELISA（Mac-ELISA）and optimised the conditions.Then we evaluated the profiles of the method.When the serum of CHIKF patient was diluted by 1: 100 in conventional manner,the 5 μg/ml CHIKV-VLP and 20 μg/ml 2H4 mAb could make the highest resolution.Futher specificity test showed that,there was no cross reaction with the serums of HFRS patients and Japanese encephalitis patients,indicating a good specificity.Futher repeatability test showed that,the detections of high OD and low OD serums were stable for four consecutive times.The repeatability of the method was good as well.We operated comparison experiment with the VLP-based Mac-ELISA method and the commercialization kit.When the VLP and the antibody were operated at the optimum concentration,9 of 10 patient serums,which are positive by PCR identification,were detected to be positive by our method.And 10 serums of healthy person were negative.While the results of commercialized Chikungunya IgM kit test showed that,10 serums of diagnosed patients were positive overall and 9 of 10 serums of healthy person were negative.Statistical analysis showed that there was no significant difference between the two groups.The sensitivity and specificity of the detection method established in this study were consistent with the commercialization kit.【Conclusion】In this study,CHIKV VLP and E2 protein were obtained by BEVS expression.The 2H4 mAb was prepared by purified E2 protein.A Mac-ELISA assay was established by CHIKV VLP and 2H4 mAb.This method was proved to be specific and repeatable.Contrast experiments with commercial kit also confirmed that the sensitivity and specificity of the detection method were consistent with the commercialization kit.Our research provides a diagnostic method for the rapid diagnosis of early infection of CHIKV,which lays an experimental foundation for further development of CHIKV rapid detection kit.