DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

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• 作者：Dan-Hui Yang ; Amina Makhmoudova ; Basil M. Arif ; Qili Feng ; Arthur Retnakaran ; Subba Reddy Palli and Peter J. Krell
• 关键词：Ecdysteroid receptor ; DNA vaccine ; Choristoneura fumiferana
• 刊名：Vaccine
• 出版年：2006
• 出版时间：12 April 2006
• 年：2006
• 卷：24
• 期：16
• 页码：3115-3126
• 全文大小：983 K

文摘

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.