副猪嗜血杆菌rfaD基因的克隆及原核表达

Cloning and Prokaryotic Expression of rfaD Gene of Haemophilus parasuis

rfaD基因编码ADP-L-甘油-D-甘露庚糖-6-异构体酶,缺失该基因会导致LOS糖链缩短和疏水性增强,从而影响细菌的致病性。为进一步探索副猪嗜血杆菌(Haemophilus parasuis,Hps)ADP-L-甘油-D-甘露庚糖-6-异构体酶的功能,本研究对Hps SC096株rfaD基因进行克隆及原核表达。根据GenBank上登录的NC_011852序列,设计引物扩增rfaD基因,获得927bp目的片段,将其克隆至pMD19-T载体。经送样测序鉴定正确后,连接到pET-32a(+)上进行原核表达,并用IPTG诱导,将诱导产物进行SDS-PAGE和Western blotting分析。SDS-PAGE结果显示,H.parasuis rfaD基因能在E.coli BL21(DE3)中表达,重组蛋白分子质量约为50ku,与预期分子质量大小一致。Western blotting分析结果表明,该蛋白质能与H.parasuis血清4型阳性高免血清产生特异性结合反应,具较好的反应原性。

ADP-L-glycero-D-manno heptose-6-epimerase encoded by rfaD gene was an enzyme essential for LOS synthesis. And it was confirmed as an important virulence factor of Haemophilus parasuis. In this study, the rfaD gene of H. parasuis serovar 4 strain clinical SC096 was amplified by PCR with a pair of specific primers based on the complete gene sequence of H. parasuis genome. And it was cloned into pMD19 T vector, then subsequently sub-cloned into expression vector pET-32a （＋）. SDS-PAGE and Western blotting analyses suggested that the recombinant BL21 （DE3） bacteria induced by IPTG were able to express the rfaD protein with molecular weight of 50 ku, which could react with the positive serum of H. parasuis sero var 4.