Eukaryotic Expression Of Neospora Caninum Jinlin Strain GRA7 Gene And The Evaluation Of Immunology

Neosporosis is the main protozoosis against the healthy development of cattle industry.The disease mainly causes abortion in pregnant cattle, stillbirth and motor system disorders in newborn calves. Currently, there are no effective methods to prevent the disease at home and abroad. Therefore, the most important issue is the screening of antigen gene and the preparation of new genetically engineered vaccine against the occurrence and development of the disease.GRA7 gene is a gene of Neospora caninum major dense granule protein which has good immunogenicity. The protein can be used as the vaccine to prevent Neosporosis. Based on the GRA7 gene sequence of Neospora caninum in Genbank (AF132217), a pair of specific primers with Xho I and EcoR I restriction sites were designed. Neospora caninum Jilin strain GRA7 gene was amplified by PCR, cloned into pMD18-T simple vector, identified by PCR and double-digestion, analysised by its sequence, compared with the published sequence in GenBank and analysised by the antigenic of its predicted protein. The correct GRA7 gene was connected with pGEX-4T-1 expression vector to construct GRA7 prokaryotic expression vector and induced in E.coli by IPTG The reactogenicity of the protein was detected by SDS-PAGE and Western blotting.The results indicated that a gene fragment which is 701bp in length was amplified, which shared 98% homology with the published sequence. The prediction of the antigenic protein showed that the antigen index of the protein expressed by the gene was high. GRA7 gene was cloned into pGEX-4T-1 expression vector. Identified by PCR and double-digestion, the prokaryotic expression plasmid pGEX-GRA7 was constructed successfully. SDS-PAGE and Western blotting showed that the molecular weight of GRA7 fusion protein is about 51 kDa, the protein can be recognized by Neosporosis positive sera and the protein has a good reactogenicity. The experiment lay the foundation for the research of Neospora caninum GRA7 genetically engineered vaccine.