Prokaryotic Expression,Purification And Monoclonal Antibody Preparation Of Ectodomain Of E Protein Of Tbev Senzhang Strain

Forest encephalitis virus,also known as tick-borne encephalitis virus（TBEV）,is transmitted by ticks and can cause forest encephalitis characterized by acute central nervous system lesions（also known as Russian spring/summer encephalitis TBE）.More than 10,000cases of forest encephalitis are reported globally each year,mainly in Eastern and Northern Europe,Russia,and also in the northeastern and northwestern forest areas of China.TBEV is associated with long-term neuropsychiatric sequelae and has no specific treatment.Therefore,TBEV infection is one of the serious public health problems.The genome of TBEV is a single-positive-stranded RNA,and its Open reading frame（ORF）encodes three structural proteins（C,Pr M,and E）and seven non-structural proteins（NS1,NS2A,NS2B,NS3,NS4A,NS4B,and NS5）.The envelope protein E is the main structural protein,which binds to host cell receptors to mediate virus entry into cells and induce protective neutralizing antibodies in the body,and is the main antigenic component of the vaccine,and the main target for serological diagnosis.The E protein consists of four main structural domains:DI,DII,and DIII,which form the ectodomain of the E protein（Envelope ectodomain,Eecto）,and DIV,which is the stem region and transmembrane anchoring region.Eecto is exposed to the surface of the virus and can be shed upon treatment of virion by trypsin.It is also known as the soluble E protein（soluble Envelope,s E）,which has a crystal structure consistent with the E protein on intact viral particles and contains almost all of the antigenic epitopes of the E protein.After the expression of structural protein Pr ME in cells,virus like particle（VLP）can be formed,which has a similar spatial structure to virus.The Reporter virus particle,prepared by transfecting Pr Me into replicant cells,RVP can be used to evaluate the neutralizing activity of serum and antibody.In this study,the main prevalent strain of TBEV Senzhang in China were selected to study.Firstly,codon-optimized Eecto sequences was chemically synthesized and a prokaryotic expression vector was constructed for expression and purification;Preparation of Eecto specific monoclonal antibodies using hybridoma technology,and evaluation of Eecto specific monoclonal antibodies based on their potency,specificity,and cross reactivity.Finally,TBEV-RVP containing the reporter gene were prepared based on the stable transformation cell line of yellow fever virus replicon constructed in the laboratory,and the neutralizing activity of the obtained monoclonal antibody was evaluated.The ralated study methods and results are as follows:1.prokaryotic expression,purification,and immunogenicity indentification of the extracellar of the TBEV-E protein.Firstly,the gene sequence of the TBEV Senzhang strain was retrieved from the Gen Bank database,and the Eecto sequence was selected for E.coli codon optimization and chemical synthesis.The Eecto gene was cloned into the prokaryotic expression vector p ET28a to construct the recombinant plasmid p ET28a-TBEV-Eecto.The recombinant plasmid was identified by PCR,and a specific band with a molecular weight of approximately 1,200 bp was amplified,which was consistent with the theoretical size;The band after double enzyme digestion was in line with the expected size;The sequencing results showed that the Eecto sequence was correct.Next,p ET28a-TBEV-Eecto was transformed into the BL21 receptor state,and induced to be expressed in small amounts by 1 m M/L IPTG.SDS-PAGE showed that the Eecto protein was expressed in the form of an inclusion body;After a large amount of induced expression,the inclusion bodies were collected by centrifugation,and dissolved in 6M guanidine hydrochloride（Gua HCl）to obtain soluble proteins under the action of refolding buffer.The latter was purified by ultrafiltration and affinity chromatographic purification by a Ni preloading column.Finally,imidazole was removed by ultrafiltration.SDS-PAGE analysis showed that high purity TBEV-Eco protein was obtained.Western blotting（Western blot）was performed using histidine labeled antibodies,The results showed that the target band was specific and consistent with the expected size.To indentify the immunogenicity of TBEV-Eecto,20μg of TBEV-Eecto protein was mixed with adjuvant,and the mice were immunized by intramuscular injection.Strengthen immunity once in the same way after the third week,and collect blood from the tail of the mouse after the fifth week.The 293T cells transfected with the TBEV-Pr ME eukaryotic expression plasmid,the above immune serum was used as the primary antibody for Western blot and immunofluorescence assay（IFA）experiments.The Western blot results showed that the specific bands were consistent with the size of TBEV-E protein,while the IFA results showed the presence of specific green fluorescence in cells expressing TBEV Pr ME.This result indicates that the serum of mice immunized with TBEV-Eecto protein can recognize eukaryotic expressed E protein,indicating that Eeco protein has good immunogenicity.2.Preparation and preliminary evaluation of monoclonal antibodies against the extracellular domain of TBEV-E proteinTo prepare monoclonal antibodies against Eecto,indirect ELISA was used to determine the serum of mice immunized with the above-mentioned Eecto protein.Two mice with the highest OD_480nm of the immune serum after 1:1000 dilution were selected to prepare spleen cells.Spleen cells and mouse myeloma Sp2/0 cells were fused with PEG 1500 as a fusion promoter.After fusion,they were selectively cultured in HAT and HT media,and the titer of the supernatant of the hybridoma cells was determined using indirect ELISA.Cells with higher titer were selected for limited dilution cloning.After 4 rounds of cloning,a total of 6hybridoma cells stably expressing Eeco protein specific monoclonal antibodies were obtained.The antibody subtypes of 2H8H4 and 2E3G6 were Ig G1 type,while 3C5D4,4H10D4,4H3B11,and 2B11H4 were Ig G2 b type.The light chains of the six monoclonal antibodies wereкChain.Subsequently,6 cell lines were injected into BALB/c mice pretreated with paraffin oil to prepare ascites and detect their titers.The results showed that4 monoclonal antibodies had a titer of 1:10⁶,while 2 were only 1:10³.In order to further analyze the specificity and cross reactivity of Eecto protein monoclonal antibodies,the ascites of six monoclonal antibodies expressing TBEV Pr ME in eukaryotic cell were used as the first antibody to carry out Western blot and IFA experiments.Western blot results showed that 2E3G6 could bind to the Linear List of TBEV-E protein,and IFA results showed that all five monoclonal antibodies except 2E3G6 could bind to the spatial conformational epitope of TBEV-E protein;Then,BHK cells were transfected with p CAGGS-YFV-Pr ME and p CAGGS-ZIKV-Pr ME,and BHK cells were infected with Japanese encephalitis virus（JEV）and dengue virus（DENV）type 2 to prepare antigen plates.The IFA results showed that 2H8H4,3C5D4,4H3B11,and 2B11H4 had cross reactivity with JEV,3C5D4,4H3B11,4H10D4 had cross reactivity with DENV2.Six monoclonal antibodies did not cross-react with yellow fever virus（YFV）or Zika virus（ZIKV）.Further affinity chromatography was performed on the ascites using protein G chromatography column,and all 5 strains except 2H8H4 were purified to obtain antibodies.SDS-PAGE showed that the obtained monoclonal antibodies had good purity.To evaluate the neutralizing activity of the Eeco protein monoclonal antibody obtained above,we used TBEV RVP to evaluate the neutralizing activity of the above antibodies.First,the p CAGGS-TBEV-C expression plasmid was constructed,and the plasmid was co transfected with the p CAGGS-TBEV-Pr ME expression vector into YFV replicon cells（constructed in our laboratory）to prepare TBEV-RVP.The purified antibody was incubated with TBEV-RVP at different concentrations at 37℃and then infected BHK cells.Finally,the relative light intensity（RLU）was measured to determine the neutralizing effect of the antibody.The results showed that none of the five monoclonal antibodies could inhibit the infection of TBEV-RVP on BHK cells.It indicates that monoclonal antibodies have no neutralizing activity.Conclusion:In this study,TBEV-Eecto protein was successfully expressed and purified in E.coli,and showed better immunogenicity.Six monoclonal antibodies were prepared,and were cross reactive with four kinds of flavivirus of the same genus.TBEV RVP was successfully prepared.This study provides a certain experimental material for the study of TBEV.