[Expression of actA gene of Listeria monocytogenes in Escherichia coli and preparation of ActA monoclonal antibodies].
The actA gene was amplified from Lm-4 strain of Listeria monocytogenes serotype 1/2a by PCR and inserted into T vector. Sequencing showed actA gene was 1833bp long and nucleotide homology was 100% compared with actA gene of Listeria monocytogenes EGD strain in GenBank. The cloned actA gene was then inserted into prokaryotic expression vector pGEX-6P-1 and pET respectively. The predicted fusion protein was detected by SDS-PAGE after IPTG induction of recombinant bacteria. The fusion protein expressed in both vectors showed approximate molecular weight of 120kDa and 97kDa. The expressed fusion protein His-ActA was purified and used as antigen to immunize BALB/c mice, hybridomas were generated with traditional hybridoma techniques. McAbs were screened by ELISA, four hybridoma cell lines secreting antibodies against ActA protein were established and the ELISA titer of these ascitic McAbs were around 1 :5 x 10(4) - 1: 1 x 10(5) . The subtype and specifity of McAbs were identified by kit and Western blot. The McAb 1A5 reacted with the expressed fusion protein GST-ActA and His-ActA in Western blot, consistent with that of mouse anti-Lm-4 polyclonal antibodies. The successful expression of ActA protein in E. coli and preparation of its monoclonal antibodies has provided useful tools for studies on the biological activity of ActA protein and its role in listerial pathogenesis.
Yin
Yue-lan
YL
Bioscience and Biotechnology College, Yangzhou University, Yangzhou 225009, China.
Dong
Hui
H
Jiao
Xin-an
XA
Jiao
Hong-mei
HM
Gu
Zhi-qiang
ZQ
Yuan
Zhou
Z
Zhang
Chen-ju
CJ
Zheng
Chao-feng
CF
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D008089:Listeria monocytogenes / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C072980:actA protein, Listeria monocytogenes