Screening Of Proteins Interacted With Theileria Luwenshuni TISP Protein With Ovine Lymphocytes By Yeast Two-Hybrid System

Theileria luwenshuni is a protozoa transmitted by ticks and parasitizein macrophages, lymphocytes and erythrocytes of small ruminants. T. luwenshuni TISP protein is a membrane protein and expressed in sporozoite, schizont and merozoite stage. The similarity was 97% of T. luwenshuni TISP and T. annulata TaSP protein at the nucleotide level. Most data showed that TaSP protein was major protein for T. annulata invading bovine lymphocytes and making it propagation without regulation. So T. luwenshuni TISP protein should hold the similar function in invading progress. Here, we used TISP protein as bait protein of the library to searching the interaction protein of ovine lymphocytes by yeast two-hybrid technique, and inferring the relationship of TISP protein with T. luwenshuni invading ovine lymphocyte through researching the function of the protein of the interaction. Therefore, the study is significant for realizing the invasion mechanism of the parasite invading lymphocyte and offer the materials for the later research.Before screening proteins by the yeast two-hybrid, the ovine lymphocytes library based on healthy ovine peripheral blood lymphocytes were prepared. The result showed that the cell density of the library was 1.08× 109个mL, the inserted fragment length is between 250-3000 bp, the titer is 1.09×108 CFU, the recombinant rate is 92%, and the library includes more than 11 kinds of protein by bioinformatic analysis.The results showed that the pGBKT7-TlSP plasmid was no auto-activation and toxicity so that it can be served as bait plasmid. The pGBKT7-TlSP bait yeast is detected the expression with the whole protein of bait yeast by Western-blotting with the first antibody of the immune sera of TISP protein from sheep, and the result showed it was expressed in the yeast and also had a good reactogenicity. Nine proteins were obtained with yeast two-hybrid library, and they were relative to ribosomal proteins and spliceosome proteins using bioinformatics analysis and mainly involved in the regulation of gene transcription, mRNA translation, cell proliferation, differentiation and apoptosis process.The screened ribosomal protein S20 from ovine lymphocytes and TISP protein were connected to the pGEX-4T-1 and pET-30a expression vector by digested with restriction double enzymes. The pET-30a-TlSP recombinant protein was purified by nickel column, and used to make immuneprecipitation test (Pull-down) with pGEX-4T-1 -S20 recombinant protein. The results showed that a protein interaction between ribosomal protein S20 and the surface proteins TISP using Western-blotting.We infer that T. luwenshuni TISP protein possesses a similar function as TaSP protein and may be involved in replication of T. luwenshuni in small ruminants.