Preparation and characterization of polyclonal antibodies against Coxsackievirus A6 VP4
10.13200/j.cnki.cjb.004730
- VernacularTitle:柯萨奇病毒A6型VP4蛋白多克隆抗体的制备及其生物学特性鉴定
- Author:
Sijin XIA
- Publication Type:Journal Article
- Keywords:
Coxsackievirus A6(CVA6);
VP4 protein;
Polyclonal antibody;
Viral particles;
Hand,foot and mouth disease(HFMD)
- From:
Chinese Journal of Biologicals
2026;39(07):807--813
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare rabbit polyclonal antibodies against Coxsackievirus A6(CVA6) VP4 and identify the biological characteristics, in order to provide core antibody materials for VP4 protein function research, CVA6 clinical diagnosis and vaccine quality control.Methods The VP4 gene was amplified by RT-PCR from total RNA of RD cells infected by CVA6 3415 strain and cloned into prokaryotic expression vector pGEX-6P-1(containing GST lable)and eukaryotic expression vector pCAGGS-HA to construct recombinant plasmids pGEX-6P-1-VP4 and pCAGGS-HA-VP4, respectively. The pGEX-6P-1-VP4 was transformed into E.coli BL21(DE3) competent cells and induced to express GST-VP4 fusion protein with IPTG. After gel cutting and purification, a female Japanese white rabbit was immunized subcutaneously on the back to generate anti-VP4 polyclonal antibodies. Indirect ELISA was applied to determine antibody titer; Western blot and indirect immunofluorescence assay(IFA) were used to detect the specific recognition ability of the antibodies to eukaryotic expression VP4 and virus VP4 in CVA6-infected cells; CVA6 virus particles(EP, FP and AP) were separated by cesium chloride density gradient centrifugation. After the morphology was confirmed by transmission electron microscope, the immunoreactivity of polyclonal antibodies against different particles was verified by Western blot.Results The recombinant plasmids pGEX-6P-1-VP4 and pCAGGS-HA-VP4 were constructed correctly as identified by double enzyme digestion. The purified GST-VP4fusion protein had a relative molecular mass of about 33 000 and a concentration of 600 ??g/mL. The titer of rabbit anti-VP4polyclonal antibodies was higher than 1∶512 000, which could specifically recognize VP4 protein expressed in eukaryotic cells and VP4 and its precursor VP0 in CVA6-infected cells. IFA detection exhibited clear and specific green fluorescence.The polyclonal antibodies could also recognize VP4 protein in FP and AP, and the signal intensity of AP was weaker than that of FP. However, only VP0 but not VP4 was detected in EP.Conclusion The rabbit polyclonal antibodies against CVA6VP4 with high titer and high specificity were successfully prepared. The antibodies are suitable for Western blot and IFA detection, and can effectively distinguish different virus particle morphology of CVA6, which provide a valuable tool for the functional research of VP4 protein and the quality control of CVA6 vaccines.
