Expression, purification and structural analysis of immunosuppressant protein, CrV1 of Cotesia rubecula (CrPDV) polydnavirus
Lihua Wei, Mario Alberto Rodríguez Pérez*
Instituto Politécnico Nacional. Centro de Biotecnología Genómica, Tel.: 0052 55 57296000 Ext: 87747. Fax: 87746. E-mails: mrodriguez@ipn.mx, weilihua8888@163.com
Key Words: AcMNPV, CrV1, transfer vector, cotransfection, Sf9
Introduction. Polydnaviruses (PDVs) are symbionts of parasitoid wasps as a natural strategy to overcome the immune defense of the Lepidopteran host. PDVs are double-stranded, circular DNA viruses found in some Ichneumonoidea families, being classified as bracovirus (BV) or ichnovirus (IV) according to the host parasitoid and viral morphology. One gene product of the BV of the parasitic wasp Cotesia rubecula, CrV1, inhibits the immune system responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. The mechanism of inactivation is still not clear. In this study, CrV1 is constructed into a baculovirus vector pAcGHLT-B and co-transfected into baculoviruses. To further understand the molecular mechanism of inactivation of host proteins by CrV1, we aim at expressing and purifying this protein in Sf9 cells to analyze the structure of the protein and perform functional motif analysis by bioinformatics methods.
Methods. We design primers and amplify CrV1 from a cDNA clone in pBluescript SK vector. Then clone CrV1 gene into a transfer vector pAcGHLT-B in frame with the glutathione S-transferase tag (GST) open reading frame (ORF). After that, using the recombinant pAcGHLT-B plasmid and AcMNPV linearized genome co-transfect into the sf9 cells. Amplify the recombinant virus in sf9 insect cells and produce recombinant protein. Then purify the protein with protein expression and purification kit. Crystallize the protein. At last, analyze the structure of the protein. pVL1392 and pAcUW31 transfer vectors with CrV1 gene are constructed using the same method.
Result and discussion. The coding sequences of CrV1 were amplified from a cDNA clone in pBluescript SK vector using rTth DNA polymerase with specific primers. The fragments were subcloned into pAcGHLT-B, pAcUW31, pVL1392 vector. After that, sequence the recombinant vectors. The recombinant baculoviruses were generated by co-transfection with a BD BaculoGlodTM Transfection Kit (BD Biosciences Pharmingen). Recombinant baculoviruses were amplified as previously described. The target protein was detected by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and purified with kit.
Conclusions and perspectives. Recombinant expression vectors with gene CrV1 and CcV1 were constructed. Recombinant viruses were obtained by co-transfection. The expression of CrV1 protein were detected in three recombinant viruses. The GST-CrV1 protein was also purified with kit. The GST tag should be cut with thrombin.
Acknowledgments. We thank SIP-IPN for financial support (project No. 20121400), and COFAA/IPN.
