| Porcine epidemic diarrhea(PED) is a kind of intestinal infectious disease that caused by Porcine epidemic diarrhea virus(PEDV), since this disease has been popular in our country, what’s more, it has caused a significant economic loss to the Pig factory. The Pig factory through daily disinfection and vaccines to prevent the disease, but under the premise of safety the traditional vaccine can’t achieve ideal effect, The study showed that the main SIg A(secretory immunoglobulin A) of piglets was the main source of the colostrum to produce the passive immune protection for PEDV. Circulating Ig G antibody in the blood can’t have effective protection on piglets, SIg A without going through the intestinal immune cannot obtain, So by the intestinal mucosal immunization is an important approach to make piglet immune protection. It is particularly important to develop a new vaccine which can be immune through the intestinal mucosa.The S protein is a structural protein encoded by the PEDV gene S, Also the major structural protein, It contains mediated virus invades a host cell receptor binding domain, also contains mediated by the body to produce antibodies to neutralize the virus antigen epitope. The COE epitope is an important epitope in the S gene, is found and named by Chang. This epitope is located in the 499-638 aa region of the S gene. Since the COE epitope is discovered, the scholars all over the world try to use the COE epitope gene to prepare the PEDV gene engineering vaccine.This experiment is to study the expression and purification of PEDV COE, which lays the foundation for the development of a new type of vaccine for porcine epidemic diarrhea virus.First of all, this experiment separate the PEDV from the pig who has the suspected swine influenza sick pigs small intestine diarrhea disease in Hennan, and then according to Gen Bank published PEDV COE sequence design out a pair of primers, Using the method of PCR AMPlification fragment length is 420 bp COE purpose and connecting the purpose fragment on the carrier PMD19- T, then transformed into Escherichia coli JM109, coated plate overnight growth, pick bacteria was cultured in liquid LB culture and AMPlification plasmid, correct identification of the evacuation company for sequencing The sequencing results and Gen Bank published the most similar to the four PEDV-COE sequence comparison, found that a mutation in the Henan local PEDV, and surrounding areas of Henan strains have some differences. Connect the COE gene fragment to PET- 32 a carrier to construct prokaryotic expression vector, The expression vector was transformed into BL21(DE3) p Lys S E.coli by the identification of correct expression. In order to explore the optimal induction conditions, We optimized the concentration and time of IPTG. The results showed that 0.6mmol/L IPTG induced by 4H can get the maximum amount of protein. We have the expression of the target protein by ultrasonic fragmentation, purified by nickel protein purification kit, and finally got the HIS-COE target protein. The successful expression of HIS-COE target protein could provide reference for the preparation of PEDV vaccine which can prevent the epidemic in the region. It also has important significance for the study and preparation of PEDV gene vaccine. |
