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Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.

ABSTRACT: The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.

SUBMITTER: Zhu Q

PROVIDER: S-EPMC4014299 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.

Zhu Qinghe Q Guo Donghua D Feng Li L Sun Dongbo D

Monoclonal antibodies in immunodiagnosis and immunotherapy 20130201 1

The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully exp ...[more]

PMID: 23600505

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Project description:Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Large-scale outbreaks of PEDV have caused huge economic losses to the pig industry since 2010. Neutralizing antibodies play a pivotal role in protecting piglets from enteric infections. However, there has been no systematic report on the correlations between neutralizing antibody titers (NTs) and absorbance values of IgG or IgA to all PEDV individual structural proteins in clinical serum, fecal, and colostrum samples. In this study, the spike protein S1 domain (S1), membrane protein (M), envelope protein (E), and nucleocapsid protein (N) of the variant PEDV strain AH2012/12 were expressed and purified by using the human embryonic kidney (HEK) 293F expression system. A total of 92 clinical serum samples, 46 fecal samples, and 33 colostrum samples were collected, and the correlations between IgG or IgA absorbance values and NTs were analyzed. R2 values revealed that anti-S1 IgA absorbance values show the highest agreement with NTs in all serum, fecal, and colostrum samples, followed by the N protein. The correlations between anti-E or M IgA and NTs were very low. However, in the colostrum samples, both IgG and IgA to S1 showed high correlations with NTs. In addition, compared with E and M, the highest correlations of IgA absorbance values were with N and S1 in serum and fecal samples. Overall, this study revealed the highest correlation between NTs and IgA to PEDV S1 protein. Therefore, the diagnostic method with anti-S1 IgA can be used as a powerful tool for assessing the immune status of pigs. IMPORTANCE The humoral immune response plays an important role in virus neutralization. Against PEDV, both IgG and the mucosal immune component IgA play roles in virus neutralization. However, which plays a more prominent role and whether there are differences in different tissue samples are not clearly reported. Additionally, the relationship between IgG and IgA against individual structural proteins and viral neutralization remains unclear. In this study, we systematically determined the relationship between IgG and IgA against all PEDV structural proteins and viral neutralization in different clinical samples and found the highest correlation between neutralization activity and IgA to PEDV S1 protein. Our data have important guiding implications in the evaluation of immune protection.

Dataset Information

Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.

Publications

Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.

Similar Datasets

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