A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two L...

Baculoviruses are arthropod‐specific, enveloped viruses with circular, supercoiled double‐ stranded DNA genomes [1]. They infect Lepidoptera (butterflies and moths), Hymenoptera (sawflies) and Diptera (mosquitoes) [2]. While many viruses are studied because of their damaging effects, the study of baculoviruses was stimulated by their potential utility to control insect pests [3]. Later, the uti...

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a reco...

The HA2 protein of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) is a WASP homology protein capable of nucleating branched actin filaments in the presence of the Arp2/3 complex in vitro. To determine the role of ha2 in the HearNPV life cycle, ha2 knockout and ha2 repair bacmids were constructed. Transfection and infection analysis demonstrated that the ha2 null bac...

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under...

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed ...

The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the ...

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacDeltaF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When H...

Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for relate...

The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line...