摘要
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38.3ku,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。
To develop SAT Ⅱ-specific FMDV serological diagnostic method,six peptides verified to be with good antigenicity 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-1 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-1.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 Ⅱ FMDV inspection.
出处
《中国畜牧兽医》
CAS
北大核心
2011年第10期55-58,共4页
China Animal Husbandry & Veterinary Medicine
基金
国家科技支撑计划(2006BAD06A14)
国家质检总局科研计划(2007IK037)资助
关键词
口蹄疫病毒
南非型
合成多肽
表达
抗原性分析
foot-and-mouth disease virus
South-Africa serotype
antigenic epitope
expression
antigenicity analysis
