Construction Of Neospora Bait Vector PGBKT7-NcGRA7 By Yeast Two-hybrid And Preliminary Screening Of Proteins Interacting With Vero Cells

Neospora caninum is parasitic on a protozoal disease caused by nucleated cells in various animals such as cattle,sheep and dogs.The necrotic lesion that occurs within a few days after the infection of the body with N.caninum leads to cell death through the proliferation of intracellular tachyzoites.Typical symptoms are miscarriage of a female animal,stillbirth,and movement disorders of newborn fetuses.The disease is a worldwide disease with a wide distribution.It has been found in Australia,Japan,South Korea and many provinces in.China,which has brought great impact on the economic development of animal husbandry.However,there is still a lack of effective vaccines to prevent Neospora.It is important to study the invasion mechanisms of Neospora and antigenic proteins for the prevention and diagnosis of Neospora.Yeast two-hybrid technology is currently widely used to study the interaction between a known protein and another known or unknown protein,with simplicity and high sensitivity.Constructing the bait vector is an important step of this experiment.Firstly,the pGBKT7-NcGRA7 vector was constructed to lay the foundation for further research on the protein interacting with the NcGRA7 gene.In this experiment,NcGRA7 gene was amplified by PCR.The recovered NcGRA7 gene was first ligated with pMD19T Simple vector and transformed into DH5a.PCR analysis and restriction enzyme digestion were performed to obtain restriction endonuclease digestion sites containing EcoRI and SalI.Point to the correct NcGRA7 gene.The pGBKT7 vector was digested with EcoRI and SalI restriction endonucleases to obtain the pGBKT7 vector containing the two restriction sites at both ends.The NcGRA7 gene containing the same restriction sites and the pGBKT7 vector were ligated and transformed into DH5a.The pGBKT7-NcGRA7 recombinant plasmids with correct PCR and restriction enzyme digestion were selected for sequencing,and were sequenced with the NcGRA7 gene sequence in GenBank 100%homology.This experiment successfully constructed the pGBKT7-NcGRA7 bait vector,which laid the foundation for the preliminary screening of NcGRA7 interacting proteins in the next step.In order to ensure that the desired results can be obtained in subsequent screening experiments,the previously constructed bait vector pGBKT7-NcGRA7 was first subjected to the self-transcriptional activity and cytotoxicity assay.The identification results showed that the constructed bait vector pGBKT7-NcGRA7 had no auto transcription activity and no cytotoxicity.The bait vector pGBKT7-NcGRA7 was hybridized with the Vero cell cDNA library to obtain 234 locus clones.Four positive plasmids,18S ribosomal RNA and 70 kDa,were obtained after PCR,plasmid extraction and transformation sequencing.Heat shock protein 8,NADH dehydrogenase subunit 1(mitochondria),oligosaccharide transferase complex.