Introduction

The baculovirus expression vector system (BEVS) has become a popular and powerful tool for expressing a variety of recombinant proteins in insect cells (1,2,3). As compared with other expression systems, the BEVS has many advantages. First, the insect cells facilitate posttranslational modification, folding, and assembly of proteins. Secondly, high expression level can be achieved. Finally, insect cell lines are suitable for suspension cultivation in serum-free condition, which allows for the production of recombinant proteins in a large scale (3,4).

The prototype baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) possessing a large circular double-stranded genome is widely used in BEVS for protein expression. In the past, the homologous recombination procedure was used for inserting foreign genes into the baculovirus genome (2). However, only 0.1% to approximately 1% of the positive recombinant baculoviruses is achieved using this method. To enhance the recombinant efficiency, several other approaches have been developed (5,6). For instance, 10% to approximately 25% recombinants can be achieved after the wild-type viral vector is linearized (7). Up to 99% recombinants can also be obtained if the ORF1629, downstream of the polyhedrin gene, is deleted completely or partially (8,9,10). Gristsun et al. (11) reported that foreign genes can be directly cloned into the viral genome by homologous recombination using appropriate primers.

An alternative approach to generate the recombinant baculovirus is the site-specific transposition with Tn7 (12). The target gene was first cloned into a transfer vector and then transposed into the bacmid genome in the presence of a helper plasmid expressing Tn7 transposase. Finally the recombinant baculovirus can be created by extraction of the bacmid DNA and transfection into Spodoptera frugiperda (Sf9) cells. Ernst et al. (13) and Lu and Miller (14) have further developed direct cloning methods for generating recombinant baculoviruses, which make it possible to construct a cDNA library using BEVS. They introduced I-SceI and Bsu36I sites into the baculovirus genome, respectively. Recently, Airenne et al. (15) presented an approach using the mutant SacB gene for promoting efficiency of selection based on the Bac-to-Bac® system (15). These methods greatly shortened the processes of obtaining recombinant baculoviruses and therefore simplified the selection procedures. With the development of BEVS, the modified baculovirus vectors carrying mammalian expression cassettes have been used for transient and stable gene delivery, not only in primary and established mammalian cells, but also in central nervous system and testes of mice (16,17,18,19).

In this work, we developed an improved direct cloning strategy to clone foreign genes directionally into the baculovirus genome and then generate recombinant viruses in a week. This is compatible with high-throughput expression technologies and is feasible in constructing a cDNA expression library. Using this method, DsRed gene and mannanase gene were cloned into the baculovirus genome and expressed in cells.

Materials and Methods

Plasmids, Bacterial Strains, and Cell Cultures

pFastBac™1, Escherichia coli DH10Bac™, and DH10B™ were purchased from Invitrogen (Carlsbad, CA, USA). pEGFP-1 and pDsRED-2 are from Clontech (Mountain View, CA, USA), and TA cloning vector pMD18-T is from TaKaRa (Shiga, Japan). pPic-man vector containing a man gene (GenBank® accession no. AF324506) encoding mannanase was constructed and stocked in this laboratory. Insect cell line derived from the ovarian tissue of the fall army worm S. frugipera is maintained in Grace's insect cell culture medium (Invitrogen) supplemented with 3.3 g yeast extract (Oxoid, Lenexa, KS, USA), 3.3 g/L enzymatic hydrolysate of lactalbumin (Difco™; BD, Franklin Lakes, NJ, USA), and 10% (v/v) fetal bovine serum (FBS; Invitrogen).

Plasmids Construction

pFGII

The gfp gene was amplified from pEGFP-1 with primers gfpF and gfpR (Table 1) by PCR. The PCR product was digested with CpoI and NotI and then inserted into the CpoI-NotI sites of pFastBac1 to obtain pFGII.

Table 1. Primers Used for Amplification and Mutagenesis (Click to enlarge)

pT-gfp and pT-mgfp

The gfp gene amplified from pEGFP-1 with primers gfpF and gfpR was cloned into pMD18-T to obtain pT-gfp. A Bsu36I site 5^′-CCTAAGG-3^′ in the gfp gene was created by using QuikChange® Site-Directed Mutagenesis kit (Strategene, La Jolla, CA, USA) with the primers mut1 and mut2. The nucleotides 5^′-GAA-3^′ at the position 135–137 bp in the gfp gene were substituted with the 5^′-AAG-3^′. The mutated plasmid was designated as pT-mgfp, and the mutation was confirmed by DNA sequencing.