Identification Of Two Anti-JEV Monoclonal Antibody Variable Region Genes, Computerized Modeling And Its Docking With JEV E Protein

Japanese encephalitis virus (JEV), a member of family Flaviviridae, genus Flavivirus,is the causative agent of Japanese encephalitis (JE). JE, also known as epidemic type Bencephalitis in China, is an acute infectious disease of the central nervous system,preferably affecting children under ten years old, transmitted by mosquito bites, andresponsible for substantial morbidity and mortality of severe cases around the world,leaving about one third of survivors with various neurologic sequela. Consistent with theglobal climate warming, the JE epidemic areas are showing trendy expansion threateningmillions of populace, and thus render a current global public health problem.Envelope (E) glycoprotein, one of the three structural proteins of JEV, constitutes themajor outer virus structure, spikes that protrude along the lipid membrane of virons, andinvolves in the process of host cell attachment, membrane fusion between the virusenvelope and host cell membrane and later virus penetration, and thus is closely associatedwith host tissue tropism and pathogenesis. In addition, E protein possesseshemagglutination activity and neutralization epitopes that elicit protective neutralizingantibodies of the hosts. In laboratory, we obtained two lines of hybridoma cells (2H4and 2F2) stably secreting anti-JEV monoclonal antibodies (McAb) with high neutralizationtiter that had been shown to be passively protective from JEV challenge on several animalmodels. However, the detailed mechanism of neutralization antibodies is not completelydefined yet. Hence, in this work, we intended to get variable region gene sequences of theMcAbs by RT-PCR, confirm their binding activity to JEV after expression in E. coli, build3D conformation of the V regions of McAbs based on the sequences, and learn thosecrucial amino acid residues pivotal to neutralization activity through molecular dockingwith the established JEV E conformation. The results will be of help for solving theneutralization of2H4and2F2against JEV infection. The details of the work are listedbelow.1. Variable region gene sequences were obtained.2H4and2F2hybridoma cells were routinely cultured, collected, the total RNA wasextracted by RNAiso Plus, and RT-PCR was performed. Primers for RT-PCR weredesigned according to the murine IgGFR1and FR4genes in GenBank. The putative Vregion gene sequences of the McAbs were analyzed by BLASTN and IMGT/V-QUEST inthe databases (GenBank+EMBL+DDBJ+PDB) to identify their FR and CDR regions. Thesequence results revealed that the V region genes of both2H4and2F2light chains wereidentical of321bp in κ type, encoding107amino acid residues with a cysteine at site92,while V region genes of heavy chains were both of354bp, encoding118amino acidresidues with the two characteristic systeines at sites22and96. All V region genesexhibited standard FR1-FR4and CDR1-CDR3of murine immunoglobulin γ subclass.2. The heavy genes of2H4and2F2were expressed and their binding activity toJEV was confirmed.To facilitate further purification, pRSET A vector with a His tag was used. The heavygene expression products were purified by nickel ion affinity chromatography and subjectto SDS-PAGE, JEV binding activity was verified by indirect ELISA.3. Computerized modelling of2H4and2F2variable regions2H4and2F2variable region genes were analyzed by BLASTP in www.ncbi.nlm.nih.gov/BLASTP, and homogenous proteins were searched out in PDB database. The three dimensional conformation with the lowest energy of2H4and2F2variable regions wereestablished by MODELLER9v1after initial modelling and further optimization.4. Molecular docking of2H4and2F2variable regions with E protein of JEVThe docking was performed by Discovery Studio v2.1, and the results revealed thatfive and nine amino acid residues of JEV E protein were recognized by2H4and2F2respectively, that is,16Ser,37Asp,353Arg,375Glu, and378Phe by2H4, while13Glu,16Ser,37Asp,300Thr,336Lys,347Asp,354Leu,387Arg, and388Gly by2F2. Indeed,more binding sites of2F2to E protein than2H4agreed with the fact that2F2possesseshigher neutralization activity than2H4does. Among all sites,13Glu,16Ser,37Asp and300Thr, located in domain I of JEV E protein as the others in domain III, are neverreported. Distinct16Ser and37Asp recognized both by2H4and2F2clearly implicatedthat they are likely to be the crucial amino acid residues in domain I pertaining to McAbneutralization activity. In addition,353Arg recognized by2H4was first unveiled,indicating that this residue may be closely associated with the uniqueanti-hemagglutination activity of2H4.336Lys of E protein recognized by2F2was quiteapproximate to the reported337Ile residue, and354Leu was first discovered.