Baculoviruses form a largest and most diverse groups of insect-pathogenic viruses. They are safe for plants, vertebrates and - what is particularly important - for mammals and humans. It was established that these insect specific viruses are widespread in nature among economically important insect pests and they may be applied in pest control programe. Numerous field trials demonstrated usefulness of baculoviruses as viral insecticides, however numerous chemical pesticides with broad spectrum and low cost of production made the natural, viral insecticides economically unprofitable on the market. Only later, at the beginning of the 1980d and 1990s it became apparent that numerous agricultural, environmental and human health problems were a consequence of a widespread use and accumulation of chemical pesticides in the environment. The necessity to develop biologically safe insecticides again focused the attention on the natural, very specific enemies of pest insect. Currently, the research concentrates on lowering the costs of growing baculoviruses in large scale. In addition, attempts have been made to improve the virulence of pesticidal baculoviruses and to expand their host range. These experiments are now in progress in several laboratories. We may expect that in the future a baculovirus genome will be modified by way of changing or deleting its important parts rather than by the expression of additional genes coding for important regulators of insect metabolism.
Baculoviruses are a diverse group of large viruses with covalently close double-stranded DNA genomes of 80-200 kilobasepairs (kbp). Baculoviruses are pathogenic for invertebrates, primarily for insects. Baculovirus particles exist in two biochemically and morphologically distinct forms, an extracellular, nonoccluded (NOV), budded virus (BV) and an occluded form (OV), which are known as polyhedral derived viruses (PDV). Baculovirus genes expression is divided into three basic phases: early (E), late (L) and very late (VL). Briefly, these phases correspond biologically to: (E) reprogramming the cell for virus replication, (L) producing BV and (VL) producing OV. The several baculovirus genes are nonessential for virus replication, and their lack in viral genome does not have any effect on forming of infectious virus particles in the tissue culture. Some of the gene expression is driven by very strong late promoters (polyhedrin and p10) and their loci are ideal cloning sites for genes of heterologous proteins. The baculovirus expression vector system is the powerful tool for production of foreign proteins. One of the major advantages of the insect cell/baculovirus system over bacterial and mammalian systems is a very high expression of recombinant proteins, which is in many cases, antigenically, immunogenically and functionally similar to their native counterparts.
Baculovirus system has been used for the expression of genes of herpesvirus glycoproteins. In this system highly glycosylated proteins are obtained. The glycoproteins may find potential use as vaccines and in diagnosis of pseudorabies.
Baculovirus vectors are very useful in producing high levels of proteins. This system yields recombinant proteins similar to their original counterparts. Expression of foreign genes occurs in infected cells which provide a suitable environment for post-translational modifications and folding of the protein product. The size of baculovirus genome allows to insert large DNA segments (up to 25kb). Constructions of recombinant baculoviruses are very simple and need minimum of viral manipulation.
The commercial exploitation of the baculovirus expression system for heterologous protein or biopesticides production requires an efficient large-scale cultivation method. This review summarized recent developments concerning the scale-up of insect cell culture and baculovirus gene expression. We described novel bioreactor systems (stirred tank bioreactor, bioreactor airlift and cell-lift, membrane bioreactor), culture modes (batch, fed-batch, continuous) and different strategies used for cell cultivation and baculovirus replication.
The recombinant human prolactin was synthesised as an extracellular protein expressed in baculovirus system. The concentration of prolactin in TC-100 medium was approximately 40 mg/l when the conditions of recombinant virus infection were properly chosen. The human prolactin present in culture medium was stable at 4C for several months up to one year. The recombinant product was a survival factor for the insect cells. In the presence of prolactin in the medium, the cells did not show any signs of lysis or disruption, which is in agreement with the view of the antiapoptotic action of prolactin. The results of Western-blot analysis showed similar ratio of glycosylated/non-glycosylated forms of the recombinant product to the hormone forms present in human physiological (osmiotic) fluids. The recombinant protein was biologically active as determined in mammary gland explant system. The recombinant hormone present in the culture media was shown to induce mRNAs for two milk proteins ? beta-casein and WAP in mammary explants cultured in the presence of insulin and hydrocortisone. The effect of the hormone was dose-dependant and the largest accumulation of both mRNAs was observed at rec-hPRL concentration of 0.1 mug/ml (approx. 4.3 x 10 -9 M). In this respect, the activity of the recombinant human prolactin was equal or even higher than that of bovine pituitary prolactin or human growth hormone.
The baculovirus expression vector system (BEVS) is a powerful tool for the heterologous protein production. The productivity of BEVS depends on: insect cell lines and their growth parameters, medium composition, multiplicity of infection (MOI), time of infection (TOI), quality of viral inoculum and scale of cultivation. The factors influencing foreign gene expression level in BEVS ? were the subject of the presented review.
JavaScript jest wyłączony w Twojej przeglądarce internetowej. Włącz go, a następnie odśwież stronę, aby móc w pełni z niej korzystać.