Prokaryotic Expression Of E^rns、NS4A、NS4B And NS5A Proteins Of CSFV And Preparation Of The Polyclonal Antibodies

Classical swine fever is widely epidemic in the world and in our country, causing great economic losses. Vaccination for long term by using live vaccines leads classical swine fever virus (CSFV) to mutate and can result in different courses of the disease, with a multi-spectrum of clinical signs. CSFV has developed strategies to evade recognition by the innate immune system of the host. The immune system fails to recognize pestiviral components as non-self and causes the immune tolerance, and consequently results in persistent infecion of animals. Ems of CSFV is capable to elicit neutralizing antibody which plays an important role in the process of persistent infection in pigs and in the process of antiviral immune response. NS4A, NS4B and NS5A proteins of CSFV are mainly involved in viral RNA replication. Four proteins Erns, NS4A, NS4B and NS5A of CSFV play a critical role in pathogenic progress. Development and production of the antibodies to specific to the four proteins of CSFV can facilitate to further study the structures and functions of Erns, NS4A, NS4B and NS5A of CSFV. In this study, our objective is that preparations of polyclonal antibodies against Ems, NS4A, NS4B and NS5A, and use them for further exploring the interaction between CSFV and host.In the study, the Erns, NS4A, NS4BΔ205-347and NS5A gene were cloned and then four genes were subcloned to the prokaryotic expression vector PET-32a(+). The recombinant expression plasmid PET-Erns, PET-NS4A, PET-NS4BΔ205-347and PET-NS5A were successfully constructed, and then the recombinant expression plasmids were respectively transformed into E.coli BL-21or E.coli BL-21derivative for inducting expression by IPTG Using SDS-PAGE and Western-blot analysis identified that Erns, NS4A, NS4BΔ205-347and NS5A proteins could highly express in the E.coli BL-21or E.coli BL-21derivative bacteria. The three proteins Ems, NS4A and NS5A were mainly expressed as a form of inclusion body. However, the NS4BΔ205-347product is confirmed as a form of solubility and/or inclusion body. These proteins in inclusion body were purified by Ni-NTA resin in reduced condition and were then re-folded to become active. The soluble protein NS4BΔ205-347was purified in non-reduced condition by Ni-NTA resin. Finally, the purified Ems, NS4A, NS4BΔ205-347and NS5A proteins were used as antigens to immunize mice of4-weeks old through rational immune procedures and the polyclonal antibodies against the four proteins were obtained.Using western blot analysis, the four polyclonal antibodies against Erns, NS4A, NS4B Δ205-347and NS5A could respectively react with their corresponding recombinant proteins, and polyclonal antibodies against NS4BΔ205-347could react with NS4B-GFP fusion proteins eukaryotic expressed in PK-15cells; The polyclonal antibody against NS5A could react with NS5A-GFP protein; The polyclonal antibody against NS4A, NS5A could respectively react with NS4A, NS5A in CSFV-infected PK-15cells. Using indirect immunofluorescent assay, the four polyclonal antibodies against Erns, NS4A, NS4BΔ205-347and NS5A could specifically react with Erns, NS4A, NS4B and NS5A proteins in CSFV-infected PK-15cells, respectivelly, and demonstrated a high sensitivity and specificity. The Erns, NS4A, NS4B and NS5A proteins of CSFV were seen in CSFV-infected PK-15cells and localized in the endoplasm.