| CD4+CD25+T cell, called natural regulatory T cell (nTreg), may be a limiting factor for the magnitude of effector responses. On the other hand, nTreg may help limit the collateral tissue damage caused by vigorous anti-infectious immune responses. Recent work has shown that the transcription factor forkhead box P3(Foxp3) was not only identified as a master regulator for the development and the function of nTreg, but also represented the most specific marker identified thus far for nTreg. Therefore, the development of a rapid and specific assay for detection of Foxp3is extremely important.According to gene of porcine Foxp3and codon usage bias in E.coli, gene sequence was optimized and synthesized. In target sequence, GC content was adjusted to57.58%. Structures inimical to gene expression were removed. Codon Adaptation Index rose from0.60to0.88, so heterologous expression level of target sequence was increased. Target sequence amplified by PCR was cloned into prokaryotic expression vector pET-28a(+), and then transformed into host strain E.coli BL21(DE3). The plasmid of positive clone was determined by restriction enzyme digestion and sequencing. Compared with the desired sequence, the results showed that the consistency was100%. The fusion protein with the size of about48kDa was induced by IPTG and purified.The rabbit antiserum against porcine Foxp3protein was obtained by immunizing a rabbit with purified fusion protein. After the last immunization, the titer of antiserum was about1:64000by ELISA. Western blotting analysis showed that the antiserum can specifically recognize the target protein. The IgGs were purified from antiserum by Protein G affinity chromatography. SDS-PAGE result demonstrated that the purified IgG consists of two bands,49.5KDa of heavy chain and26.9KDa of light chain.Spleen cells from BALB/c mice immunized with purified fusion protein were fused with SP2/0myeloma cells after last immunization. Hybridoma supernatants were screened for the specific antibodies by indirect ELISA. Five hybridoma cell clones (2A4,2C4,3F3,4C8and4C11) were successfully obtained, which can be cultured stably and secret McAb against porcine Foxp3protein. The Ig isotypes of all McAbs were IgGl,κ. The ELISA titers of ascites and supernatants were3.2×105,1.6×105,3.2×105,1.28×106,6.4×105and4.0×103,10×103,8.0×103,4.0×103,2.0×103, respectively. The results of ELISA and Western blotting indicated that the five clones of McAb can specifically react with porcine Foxp3protein from PBMC and fusion protein. SDS-PAGE result also demonstrated that the purified IgG consists of two bands,49.5KDa of heavy chain and26.9KDa of light chain.A sandwich ELISA detecting porcine Foxp3protein was developed. The test conditions were optimized by reagent titration. The purified McAb and the purified PcAb were used as coating antibody and detecting antibody, respectively. The optimal concentration for McAb was1.25μg/mL and for PcAb0.9375μg/mL. The optimal dilution rate of enzyme labeled antibody was1/6000. There was no cross-reaction between the various reaction components. Minimum detectable concentration was0.458ng/mL. The linear range of the dose-response curves was117.19-7.32ng/mL. A least-squares regression analysis supports a simple linear dependence of OD on Foxp3concentration. The relationship was OD=0.0106[Foxp3]+0.1387with an R2(coefficient of determinant)=0.9981. The within-run and between-run coefficients of variation were4.89%and14.26%. According to clinical symptoms, samples divided into disease and disease-free group. The two groups were the normal distribution. The sample size, mean and standard deviation of the two groups were17,108.82,85.77and27,167.54,89.97, respectively. One-way ANOVA analysis showed that the difference was significant (p<0.05).The successfully developments of McAb and PcAb against porcine Foxp3and indirect sandwich ELISA for detection of porcine Foxp3provide a favorable means and tools for further study of porcine Foxp3and nTreg. |
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