Polyhedrin -Negative BmNPV Bac to Bac

Before innovation, the original silkworm expression system was not only difficult to recombine with foreign fragments, but also had difficulties in virus amplification. To solve the problem, improvements had been made based on the process of AcMNPV Bac-to-Bac construction. The novel approach cloned a large fragment (Figure 1.) containing:

• low-copy-number mini-F replicon
• a Kanamycin resistance sequence
• a lacZ^a peptide segment
• a bacterial transposon target site mini-attTn7 that located at the N-terminus lacZ^a gene into polyhedrin locus of BmNPV genome to replace the polyhedrin, which is an unnecessary gene for baculovirus replication but essential for viral capsid

Figure 1. Structure of BmNPV Genome after Manipulation (BmBacmid)

Essential Component of Polyhedrin-Negative BmNPV Bac to Bac System

The manipulated BmNPV genome is named as BmBacmid and then is transferred into Escherichia coli DH10 (E. coli DH10BmBac). The novel established Polyhedrin-Negative BmNPV Bac to Bac system generally consists of:

• A donor plasmid which carries the interest fragment (Figure2 A)
• The E. coli DH10BmBac which hosts BmBacmid and contains a helper plasmid (Figure2 B),
• The polyhedrin-negative BmBacmid (Figure2 G) 

This system mainly relies on Tn7 site-specific transposition between donor plasmid and BmBacmid to generate recombinant baculovirus.

Figure 2. The Expression Process of Polyhedrin-Negative BmNPV Bac to Bac System

Why Choose Polyhedrin-Negative BmNPV Bac to Bac System

Polyhedrin-Negative BmNPV Bac to Bac protein expression process is so sample that it only takes 3-4 weeks to obtain the target protein. Especially suitable for customers who requires small-scale and fast expression.

• Highly infectious. Can infect hemocoel tissues and cultured cells due to the knocking down of polyhedrin.
• Low cost. Silkworm larvae has large body and low rearing cost.
• Simple step. Construct recombinant BmBacmid then inject to larvae, no need for transfection or virus amplification.
• High yield. The strong polyhedrin promoter is remained and is used to enable the high level of target protein expression.
• Time saving. The recombinant BmBacmid can be constructed within a week and harvest in 2-3 weeks after injection.

Our services for Polyhedrin-Negative BmNPV Bac to Bac

• Subclone the gene of interest into donor plasmid between the two Tn7 site, downstream of the polyhedrin promoter
• Transform the recombinant donor plasmid into E. coli DH10BmBac for transposition
• Colony selection by x-gal culture medium
• Extract the recombinant bacmid from E. coli DH10BmBac and inject the bacmid into the silkworm larvae
• Recovery of protein from hemolymph or fat body

Relevant service

Polh-positive BmBacmid: 

Polyhedrin remained. Highly infectious to the midgut epithelial cells and mediate infectious to larva transmission. Requires transfection but not amplification. Suitable for large-scale protein production.

Creative BioMart’s deeply developed research platform endows it with expertise in gene manipulation. We are always committed to improving the existing system to maximize its advantages. Browse our service instrument to decide a suitable BmNPV BEVS expression approach for your protein or contact us for help. If you had already made your own expression plan which is beyond our list, our edge-cutting technology platform still able to provide assistance for you. All you need to do is to contact us and put forward your requirements.