In order to achieve high-efficiency expression of the porcine circovirus type 3 Cap protein in recombinant Escherichia coli,dissolved oxygen(DO)control strategy,oxygen uptake rate(OUR)control strategy and combined control strategy of DO and OUR were selected to investi-gate their effect on the expression of target protein in a 10 L fermenter.The high-density fermenta-tion process of recombinant E.coli was determined by investigating the best control range of OUR.The recombinant protein was identified by SDS-PAGE and Western blot,and the immunoge-nicity of the protein was evaluated by the animal experiment.The results showed that when the DO control strategy and OUR control strategy were used,the expression of target protein was low.Acetic acid was the key factor affecting the expression of target protein.When acetic acid concen-tration reached 1.02 g/L,Cap protein yield was reduced by 55.8％.In order to reduce the produc-tion of acetic acid,DO was selected to control oxygen supply before feeding,and OUR was selected as the parameter to control oxygen supply after feeding.When OUR value was maintained at 140-160 mmol/(L·h),the Cap protein yield was up to 650 mg/L.Western blot analysis confirmed that PCV3 Cap possessed antigenicity and specificity.Animal experiment showed that the antibody pos-itive rate was 100％after the second immunization,indicating that the target protein had better immunogenicity.The high density fermentation controlled by the combined control strategy of DO and OUR could achieve efficiently soluble expression of PCV3 Cap in the fermenter,which laid the foundation for the vaccine development.

In order to achieve high-efficiency expression of the porcine circovirus type 3 Cap protein in recombinant Escherichia coli,dissolved oxygen(DO)control strategy,oxygen uptake rate(OUR)control strategy and combined control strategy of DO and OUR were selected to investi-gate their effect on the expression of target protein in a 10 L fermenter.The high-density fermenta-tion process of recombinant E.coli was determined by investigating the best control range of OUR.The recombinant protein was identified by SDS-PAGE and Western blot,and the immunoge-nicity of the protein was evaluated by the animal experiment.The results showed that when the DO control strategy and OUR control strategy were used,the expression of target protein was low.Acetic acid was the key factor affecting the expression of target protein.When acetic acid concen-tration reached 1.02 g/L,Cap protein yield was reduced by 55.8％.In order to reduce the produc-tion of acetic acid,DO was selected to control oxygen supply before feeding,and OUR was selected as the parameter to control oxygen supply after feeding.When OUR value was maintained at 140-160 mmol/(L·h),the Cap protein yield was up to 650 mg/L.Western blot analysis confirmed that PCV3 Cap possessed antigenicity and specificity.Animal experiment showed that the antibody pos-itive rate was 100％after the second immunization,indicating that the target protein had better immunogenicity.The high density fermentation controlled by the combined control strategy of DO and OUR could achieve efficiently soluble expression of PCV3 Cap in the fermenter,which laid the foundation for the vaccine development.

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology

Objective To analyze the differential expression of Stathmin in human cells infected with human-tropic porcine endogenous retrovirus(PERV)and to explore the potential molecular effect of human-tropic PERV on human cells.Methods HEK293 cells were infected with the human-tropic PERV infectious molecular clone.PCR,real-time RT-PCR and immunofluorescence analysis were applied to confirm that HEK293 cells were infected.Then real-time RT-PCR and Western blot were carried out to analyze the differential expression of Stathmin at the mRNA level and protein level,respectively.Results HEK293 cells were infected by human-tropic PERV.Real-time RT-PCR and Western blot analysis showed that Stathmin was up-regulated in HEK293 cells infected with PERV compared with the control cells.Conclusion Stathmin was up-regulated in HEK293 cells infected with human-tropic PERV.These studies will be helpful for revealing the interaction of PERV and human cells,and for understanding the molecular effect of humantropic PERV on human cells.In addition,it suggested that PERV infection may infect cell growth and physiological functions,even be pathogenic.These will help to clarify the biologic characteristics of PERV and evaluate the safety of PERV in pig to human xenotransplantation.