To develop SAT FJ -specific FMDV serological diagnostic method, six peptides verified to be with good antigeni-city in the former study were linked by flexible amino acid linker GS or PPPS, the related nucleotides were synthesized and expressed in vitro by cloning into vector Pgex-6P-l and inducing with IPTG. The specificity of the expressed GST fusion protein was examined by SDS-PAGE and Western blotting analysis using anti-GST sera and antiserum against SAT II FMDV. The fusion protein was purified by GST affinity chromatography purification system and excised on column. The antigenicity of purified protein VP1 - VP3 was examined using the developed indirect ELISA assay. PCR and DNA sequencing results showed that the gene VP1- VP3 had been integrated accurately into vector Pgex-6P-l. SDS-PAGE showed that the fusion protein was 38. 3 ku in size and existed in the form of insoluble inclusion body. Western blotting analysis results indicated that the fusion protein was with good specificity. Indirect-ELISA results showed that the VP1- VP3 protein had good antigenicity. The successful expression of mass-peptide will lay the foundations for the latter establishment of serological diagnostic methods for SAT D FMDV inspection.%在对南非Ⅱ型口蹄疫病毒抗原分析基础上,将已经筛选出的6条抗原性良好的多肽采用柔性linker拼接,人工合成相应核苷酸后连入T载体中.将酶切回收的串联基因( VP1—VP3)克隆于表达载体pGEX-6P-1中,获得重组质粒pGEX-VP1- VP3.该质粒转化于感受态细胞BL21 (DE3) plysS中,经IPTG诱导后进行了可溶性分析和Western blotting分析,且对融合蛋白柱上酶切纯化后进行了抗原性分析.重组质粒pGEX-VP1—VP3的PCR和测序结果表明,VP1—VP3串联基因已成功插入pGEX-6p-1载体中;pGEX-VP1—VP3融合蛋白分子质量约为38.3 ku,并以包涵体形式存在；Western blotting结果显示,该融合蛋白与南非Ⅱ型FMDV阳性血清能发生特异性反应；酶切纯化后蛋白间接ELISA鉴定结果表明,表达的VP1—VP3蛋白具有良好的免疫原性与反应原性.串联多肽的成功表达,将为南非型口蹄疫血清学检测方法建立奠定基础.
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