NS4A protein of Zika virus influences the neuronal migration of mouse cortex / 基础医学与临床

Objective To determine the effect of NS3 and NS4A proteins of Zika virus on the neuronal migration in vivo.Methods To identify the coding sequence of NS3 and NS4A,the genome of Zika SZ01 was sequenced by rapid amplification of cDNA ends (RACE) and reverse-transcription PCR,then NS3 and NS4A was constructed in pCIG vector fused with Flag-tag to express these proteins.And then these plasmids was transfected into the embryo brain of E13.5 mice by in utero electroporation,the distribution of the cells which express these proteins in the cortex was detected by Flag,eGFP and TBR1 fluorescence in E18.5 mice through immunohistochemistry so as to assess the influence of viral proteins on the neuronal migration of mouse cortex.Results 1) Sequence results showed that the amino acid sequence of NS4A is consistent with NCBI data,while NS3 has 1 amino acid mutant.2) As the fluorescence of Flag and eGFP can co-localization,the eGFP fluorescence signal marks the cells that have expressed these virus proteins in cortex.3) TBR1 fluorescence shows the distribution of the cells that express NS4A in vivo are significantly different from pCIG control and NS3 (P＜0.001).Conclusions The NS4A protein of Zika virus may affect the neuronal migration in vivo.

Cloning and expression of Dengue virus nonstructural protein 4A gene and affinity purification of its interacting proteins / 中华微生物学和免疫学杂志

Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.

SCREENING AND IDENTIFICATION OF ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENT AGAINST HCV NS4A / 解放军医学杂志

To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus NS4A(HCV NS4A)so as to lay a foundation for developing anti Id scFv vaccine againist the hepatitis C virus. The recombinant phage antibody library was panned by hepatitis C virus NS4A monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning, 82 clones specific to HCV NS4A antibody were determined with the enzyme linked immunoadsorbent assay(ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competitive inhibition assay. The DNA sequence of the positive clone was determined. The result showed that HCV NS4A anti Id scFv had a specific combination character with hepatitis C virus NS4A monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 789 bp. The results suggested that the anti Id scFv fragments to HCV NS4A monoclonal antibody could be successfully selected by recombinant phage antibody library screening technique, which might pave a way for the study of new preventive and therapeutic strategy of hepatitis C using anti Id scFv.