Amplification of Autographa californica Nuclear Polyhedrosis Virus Polyhedrin Gene Leader Sequence by Polymerase Chain Reaction for Constructing Baculovirus Vectors

By means of a selective DNA amplification technique, the polymerase chain reaction (PCR), a 90 bps DNA fragment containing a normal length leader sequence of AcMNPV polyhedrin gene was amplified with new sequence information of EcoR I site at its 3' end.

The 90 bps fragment was cloned to pUC18 Sma I site and sequenced. The fragment with correct leader sequences was then excised from pUC18 using EcoR V site at 5' end and the newly added EcoR I site at 3' end.

A new Baculovirus transfer vector with a normal length leader sequence was constructed from previously constructed pAcBsd100-22HHd113, which contains an extended leader sequence with an excess of 22 nucleotides at its 3' end, with EcoR I/EcoR V fragment synthesized by PCR.

Leader sequence with point mutation was also synthesized by PCR. With the same cloning strategy, the second new transfer vector with mutated leader sequence was constructed. Hepatitis B virus surface antigen gene acted as reporter gene to investigate the influence of leader sequences on the level of foreign gene expression.