Abstract:

In order to acquire polyclonal antibody of high titer and specificity against PR-1a, 1 polypeptide with sequence-specific were obtained by Blastn, Blastx and Expasy software based on the primary structure of information about PR-1a of tobacco in NCBI GenBank. The polypeptide was synthetized by fmoc solid phase synthesis methods, and its purity and molecular weight were also determined by HPLC and LC-MS, respectively. The purity value and molecular weight reached at 93.2% and 2088.17, respectively. The polypeptide was coupled with keyhole limpet hemocyanin (KLH) by way of EDC. Anti-sera were acquired by immunizing rabbit with Pep-KLH emulsified by Complete Freund’s adjuvant (CFA) and Incomplete Freund’s adjuvant (IFA), and polyclonal antibody was purified by affinity chromatography. The titer and specificity of anti-sera and polyclonal antibody were determined by indirect-ELISA and Western blotting with the OD value reaching at 1.0 at the dilution of 1:32000 against anti-sera; The results from Western blotting showed that anti-sera and polyclonal antibody could detected the specific band with molecular weight of 18 kD in Nicotiana tabacum K-326 leaves, the results was accord with molecular weight which was the predicted. Benzothiadiazole (BTH), a class of inducers of systemic was sprayed on leaf of Nicotiana tabacum K-326 at the 500 μg/mL, and then the total protein from leaf was extracted at the interval of 0, 6, 12, 24, 48, 96, 144 and 192 h. The results indicated that PR-1a was up-regulated at the 48 h by indirect-ELISA assays. Meanwhile, RT-PCR of semi-quantity was also used to verify the expression trends about PR-1a, the results indicated to become a similar expression trends with that of indirect-ELISA. The results shown the PR-1a polyclonal antibody reached high sensitivity and specificity, and was used to the action mechanism of inducer of Systemic acquired resistance (SAR).