关键词: 环形泰勒虫, TA04380蛋白, 原核表达, 特异性, ELISA方法

Abstract:

Objective To express recombinant TA04380 protein (rTA04380) of Theileria annulata, establish an enzyme-linked immunosorbent assay (ELISA)-based detection method and evaluate its value of application. Methods Bioinformatics analysis of TA04380 protein was performed with TMHMM and SignalP, primers were designed according to the nucleotide sequence of T. annulata TA04380 published in GenBank, and the truncated fragment (1-636 bp) of TA04380 gene was amplified by PCR to construct pET30a(+)-TA04380 plasmid and was transformed into Rosetta (DE3) competent cells, from which the rTA04380 protein was expressed by induction and then purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify the expression of the recombinant protein. The reactivity of the rTA04380 protein and its cross-reaction with other bovine piroplasma were analyzed by Western blotting. The rTA04380 protein was used as the target antigen to establish an ELISA method for the detection of serum antibodies against T. annulata; the optimal antigen coating concentration, serum and secondary antibody concentrations and the time for optimal blocking were determined by screening tests. The established ELISA method was used to detect 56 standard T. annulata negative bovine sera and 76 standard T. annulata positive bovine sera to determine the threshold value. The reproducibility and robustness of the ELISA method in the first, second and third weeks after the rTA04380 protein was coated. A total of 125 bovine serum samples were detected with the established ELISA, and compared with the reported ELISA method based on sporozoite surface antigen (SPAG) protein, the coincidence rate was calculated, and 571 bovine serum samples from 9 provinces were detected by the established ELISA method to evaluate its application value. Results The bioinformatics analysis showed that the T. annulata TA04380 protein has no signal peptide sequence, there are four transmembrane regions in the 212-413 aa region, and the amino acid sequence identity with T. orientalis homologous protein is 55%. The PCR amplification fragment length was 636 bp. The SDS-PAGE showed that the rTA04380 protein mainly existed in the inclusion bodies, and the purified TA04380 protein band was located at a relative molecular weight (M_r) of 31 940; Western blotting analysis showed that the rTA04380 protein could react both with the anti-His monoclonal antibody and T. annulata positive serum, but no cross-reaction was observed with T. orientalis, T. sinensis, B. bovis, B. bigemina reactive sera and healthy bovine serum. The optimal conditions of the ELISA method based on rTA04380 protein were: antigen is coated at a concentration of 10 μg/ml, and blocked with 1% bovine serum albumin for 1 h followed by addition of 1 ∶ 100 diluted serum and incubate for 1 h. The rabbit anti-bovine horseradish peroxidase (HRP)-IgG was added as a secondary antibody at a concentration of 1 ∶ 6 000 and incubated for 1 h. The detection threshold was 16.9%, the sensitivity and specificity were 93.4% and 96.4%, respectively, and the false positive rate was 3.6%. The positive rates of the established ELISA method and the ELISA method based on SPAG protein were 54.4% (68/125) and 49.6% (62/125), respectively, and the coincidence rate between the two methods was 85.6%. a The positive rate of 571 bovine serum samples from 9 provinces was 43.1% (246/571) using the established ELISA method. The positive rate from high to low is Jilin (85.7%, 24/28), Ningxia (54.2%, 13/24), Guizhou (50.0%, 13/26), Henan (8/16), Gansu (43.8%, 57/130), Inner Mongolia (42.2%, 35/83), Liaoning (41.1%, 30/73), Yunnan (38.2%, 42/110) and Qinghai (29.6%, 24/81). Conclusion The rTA04380 protein was successfully expressed, and the established ELISA based method presents high sensitivity and specificity, good reproducibility and coincidence, impliying potential value for wide application in the field.

Key words: Theileria annulata, TA04380 protein, Prokaryotic expression, Specificity, ELISA method