| Chikungunya virus,an arbovirus,causes chikungunya fever,which can further develop into acute and chronic debilitating arthritic disease,and is a zoonotic disease.The acute phase of infection presents with sudden onset of high fever(>39°C)with erythematous maculopapular rash,myalgia and arthralgia,and the chronic phase with persistent or recurrent arthritis,which can last from several months to several years.Approximately 50-60%of CHIKV-infected patients are reported to develop chronic disease,with an economic burden estimated at￡25,000per patient per year.There is an urgent need to establish early and rapid diagnostic methods.Although RT-PCR-based diagnostic methods are particularly unique,they are also limited by laboratory equipment,specialized technicians,and other factors.However,antigen detection,with its advantages of high specificity,rapidity,and convenience,is once again gaining widespre ad attention in the detection of emerging infectious diseases.The geographical location and ecological environment of Yunnan are extremely suitable for the survival of Aedes albopictus,and Yunnan is at risk of cross-border importation of CHIKV due to its borders with high-risk areas of Laos and Myanmar,where CHIKV,DENV,and ZIKV are all arboviruses with cross-infection,co-infection,and similar clinical symptoms in endemic regions.As a result,the situation of chikungunya epidemic and control in Yunnan Province is not optimistic.Currently,there is no CHIKV antigen test approved for clinical testing in China.Therefore,the development of CHIKV antigen detection methods has important application value.The main findings and conclusions of this thesis are as follows.(1)Expression and purification of rE2 protein.The chikungunya virus E2(1-1038 bp)gene was used as the target gene and successfully cloned into p ET28a.The rE2 protein was successfully expressed in Rosetta(DE3)after induction by isopropylthio-β-D-galactosidase.The protein was purified using His-tagged affinity resin,and the rE2 protein was verified by SDS-PAGE and Western Blot.The results showed that a protein of 40 k Da size was successfully obtained,which laid the foundation for animal immunization.(2)Obtaining monoclonal antibodies.A total of three antibody cell lines were obtained by screening stable cell lines using the hybridoma technique and named as 1B7-H7,2A2-E10 and 4D3-E8.monoclonal antibody ascites was affinity purified by Protein A and verified for specificity,antibody isotype,neutralizing activity and other properties.The results showed that:(a)The three monoclonal antibodies obtained had no in vitro neutralizing activity(b)All three monoclonal antibodies recognized rE2 protein well by ELISA,IFA and Western Blot tests.Meanwhile,they had no cross-reactivity with four serotypes of DENV and ZIKV with 100%specificity.These antibodies have good serological diagnostic potential and provide experimental material for the subsequent establishment of CHIKV antigen detection methods.(3)Establishment of the DAS-ELISA assay.The activity of the three monoclonal antibodies was verified by direct ELISA after HRP coupling,and the results showed that 2A2-E10(HRP)had the best activity.Therefore,2A2-E10(HRP)was utilized as the detection antibody,and rabbit polyclonal antibody-coated ELISA plates were used to specifically capture the antigen.The DAS-ELISA assay was established and fully evaluated.The experimental results indicated that the DAS-ELISA assay has good specificity and the lower limit of detection is 1×10~6 PFU/m L。In conclusion,this thesis established a DSA-ELISA antigen detection method,which can be used as a high-throughput screening method for early CHIKV infection,providing a strong guarantee for effective epidemic control and timely treatment,and meeting the current demand for early diagnosis of CHIKV in Yunnan Province and even in China. |
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