猪胸膜肺炎放线杆菌荚膜多糖基因的原核表达及其产物的细胞毒性

Prokaryotic expression of capsular polysaccharide genes of Actinobacillus pleuropneumoniae and cytotoxicity assay of expressed products to porcine neutrophil

参考GenBank中猪胸膜肺炎放线杆菌(App)血清2型荚膜多糖基因的序列分别设计了3对特异性引物,通过PCR分别扩增了CpsA、CpsB、CpsC 3个基因,获得长约1 137、429、1 146 bp的片段,将其分别克隆到pMD 18-T中,经酶切鉴定和序列分析获得了阳性克隆子;再将CpsA、CpsB、CpsC分别插入原核表达载体pGEX-KG后,转化BL21,在IPTG诱导下获得高效表达,经Western-blotting检测证实,表达产物CpsC有生物学活性,CpsA、CpsB无活性;将3种表达产物等量混合,做10倍递进稀释后,与分离出的猪嗜中性粒细胞作用,37℃5、0 mL/L CO2培养箱中温育4 h后加入底物液,测定D492 nm值。结果表明,App荚膜多糖对猪嗜中性粒细胞无毒性作用。

CpsA, CpsB and CpsC genes of Actinobacillus pleuropneumoniae serotype 2 were amplified with the 3 pairs of primers designed on the basis of the 3 genes sequences in GenBank by PCR. The respective amplified DNA fragments 1 137, 429 and 1 146 bp in length were cloned into pMD 18 T. After being identified by BamH Ⅰ or EcoR Ⅰ and being sequenced,the CpsA, CpsB and CpsC genes in pMD 18-T were ligated into an expression plasmid pGEX-KG. Three recombinant expression plasmids KG-CpsA, KG- CpsB and KG-CpsC were constructed and then identified by BamH Ⅰ or EcoR Ⅰ . The CpsA, CpsB and CpsC proteins expressed in E. coli BL21 were detected by Western-blotting. Different amouts of the expressed CpsA, CpsB and CpsC mixture and an empty expression vector as control interacted with extracted porcine neutrophils and D492nm values were detected. The result showed that the mixture had no cytotoxicity to the porcine neutrophils.