Neospora Caninum GRA7 Gene And GRA2 Gene Construction And Expression Of Gene Tandem Vector

Neosporosis is a world-wide parasitic disease caused by Neospora caninum,which belongs to Phylum Apicomplexa,Sporozoa,Coccidiasina,Eucoccidiorida,Sarcocystidae and Neospora.It is mainly parasitic on the central nervous system,muscle,liver,brain and internal organs of the host.This disease was firstly found in Norway in 1984,and Dr.Dubey named it Neospora caninum in 1988.The final host of Neosporosis are canine and fox,and the intermediary host are cattle,goat,horse,pig and rabbit,etc.This disease mainly caused abortion or stillborn fetus of pregnant animals,and the dysfunction of the motor nerve in newborn babies.It has caused damage in different animals,especially causes great damage to cattle.It has been one of the main reasons giving rise to the abortion of cattle.The disease widely distributes in Europe and 30 more countries with a world wide distribution.The positive rate of serum antibody in some of the cattle herd can be up to 80%,and in our country,the positive rate of serum antibody in dairy cattle is about 30%.As a result,the pressing matter of the moment is to make neosporosis under validation control.In the trial,the dense granule protein GRA7 gene and GRA2 gene of N.caninum were combined by flexible amino acid,and were made into a series by SOE PCR technique,and were linked into the expression vector,so that it could express a recombination protein with a good immunogenicity.The total RNA was extracted from the cultured N.caninum Nc-1 strain in the trial,and was made into cDNA by reverse transcription.The N.caninum cDNA was used as a templet,and the primer of NcGRA7 was used to amplify for the first time,and the product was a target fragment of 696 bp;the primer of NcGRA2 was used to amplify for the second time,and the product was a target fragment of 567 bp;by SOE-PCR,the two products of PCR was combined and attached together,and the final product was serial gene NcGRA7-NcGRA2 of 1263 bp.Then the serial gene was combined with pMD-19-T sinple vector.Identified by PCR,double enzyme digestion and sequencing,the two genes were confirmed to be in a tandem successfully.Then the NcGRA7-NcGRA2 gene fragment was combined with the pronuclear expression vector pGEX-4T-1,and the result of Western blot showed that,the recombination pronuclear expression vector successfully expressed the serial protein.The NcGRA7-NcGRA2 gene fragment was attached to the eukarya expression vector pcDNA3.1 and identified by IFAT,the result showed that the recombination eukarya vector could express exogenous protein in mammal cells.