Prokaryotic Expression Of PCNA Gene Of Theileria Annulata And Establishment Of Indirect ELISA

Theileria annulata disease,also known as tropical theileriasis,is a blood protozoan disease caused by tick,which parasites in monocytes and lymphocyte.Theileria annulata belongs to Apicomplexa,Piroplasmea,Theileriidae,Theileria.The clinical symptoms of this disease are high fever,anemia,body surface(systemic)lymphadenopathy,systemic hemorrhage,weight loss,fever,thrombocytopenia,leukopenia,pulmonary edema and pleural effusion,and the adventitia of the kidney is easy to peel off.According to statistics,about 250 million cattle are threatened by this disease every year in the world,and the loss caused by the disease is about 800 million dollars.It is listed as a type B disease by the International Bureau of Epidemiology,and a second type disease in China,which has a huge impact on the cattle industry.Proliferating cell nuclear antigen(PCNA)is a protein expressed in proliferating cell,which is related to the activity of tumor cells.The transformed cells by T.annulata have the characteristics of limted proliferation.According to its function and bioinformatics analysis,the protein has potential development value.In this experiment,the Theileria annulata PCNA gene was used as the research object and the PCNA protein was analyzed by using bioinformatics software.PCNA protein was induced by prokaryotic expression and purification.Then,the purified PCNA protein was used as the coating antigen to establish an indirect ELISA method.It laid the foundation for the establishment of better detection methods in the future.1.Cloning and bioinformatics analysis of PCNA gene of Theileria annulataExtracting RNA and reverse transcribing it into cDNA,the PCNA gene was cloned from cDNA,and then confirmed by sequencing.Using bioinformatics to predict and analyze the PCNA gene,the results show that the isoelectric point of PCNA protein is 4.84,which is a hydrophilic acidic protein,which contains 22 serine phosphorylation sites and 18 threonine phosphorylation sites;in PCNA the secondary structure of the protein,a-helix(h)accounts for 32.48%,β-sheet(e)accounts for 26.64%,β-turn(t)accounts for 3.65%,and random coils(c)accounts for 37.23%.It is found in the tertiary structure that the protein has no ligand and assumes a trimer state.The prediction of PCNA protein antigen sites showed that there were 8 B cell antigen sites.2.Prokaryotic expression,purification and reactogenicity analysis of PCNA proteinThe expression vector pET-30a-PCNA was successfully constructed,and the expression vector was introduced into BL21 competent cells to explore the induction time and inducer concentration,and determine the best induction conditions.According to SDS-PAGE,the expression form of PCNA protein is inclusion body with a size of 33kD.Western blot proved that the PCNA protein could only react with the positive serum of Theileria annulata,but had no cross-reaction with positive serum of T oriental,T.sinsensis and Babesiosis,indicating the protein has good reactogenicity.3.The purified PCNA protein was used as the coating antigen and explore the best coating conditions to establish an ELISA method.The coating concentration of the recombinant protein was 100ng/ml,and the best ratio of the primary anti-serum was 1:80,in the ELISA plate Incubate for 2h,the best blocking time for blocking solution was 1h,the best incubation time for adding primary antibody serum was 1h,the dilution ratio of the enzyme-labeled secondary antibody was 10000 times,the best secondary antibody incubation was 1.5h when the OD450 was close to 1.0,and the positive OD450/the negative OD450 ratio was the largest.The method of estabilishment has good specificity and sensitivity.The determination of OD450>0.197 was positive,when OD450<0.166 was negative.The sample with OD450 between the two is suspicious and needs to be tested again;intra-assay coefficient of variation and inter-assay coefficient of variation both were less than 10%,indicating good reproducibility;The positive rate was 14%and the coincidence rate was 100%.It indicated that the coincidence rate of indirect ELISA was very good.