Table of Contents
Abstract.
Use of BEV system for production of BV in insects is widely used in commercial production processes. The method is aimed in the manufacture of insecticides or in expression of medically important foreign genes. Most common strategy employed in modification of B.V is the transfer of a vector and viral DNA that are then co-infected into insect cells by use of host enzyme homologous recombination. In this research, a rapid method to extract B.V DNA for PCR analysis from cultured insect cells was used. Three steps, SDS lysis, Chloroform extraction and ethanol precipitation were employed to isolate viral DNA. The isolated DNA from viral culture was then analyzed by PCR.
Introduction
Baculovirus Expression Vector (BEV) system was developed by Summers and Smith (1987).The system enables production of biological and large amounts of recombinant proteins within a short while. In pilot scale production and use of recombinant proteins, the method is the most preferred. (Vihko 1993). This system is based on BV families, which are double stranded DNA viruses that infect vertebrates. (Matthews 1982 p106).
The advantage of this system is that there is reduction in essay from a period of days to just few hours.
Besides the method is convenient in detection of BV in cell culture, contamination, monitoring mixed viral infection, and configuration of viral genomic integrity. (Christina and Romanowski 2008, p351). The Baculovirus expression vector system (BEVS) has proven to be a powerful technology for expression of recombinant proteins in eukaryotic cells. The BEVS has many advantages that include the capacity for large DNA insertions and the capacity for simultaneous expression of multiple genes. ( Hopkins and Esposito 2009).
Results
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Comparison of virus stock titers determined using end-point dilution assays with the Sf-9 Easy Titer cell line vs. the standard Sf-9 cell line |
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Virus stock |
Sf-9 Easy Titer cell line A. |
Standard Sf-9 cell line B. |
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1 |
2.0 × 108/mLc |
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2 |
7.5 × 108/mL |
7.5 × 108/mL |
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3 |
8.8 × 108/mL |
8.8 × 108/mL |
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4 |
1.1 × 108/mL |
9.7 × 107/mL |
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5 |
2.1 × 108/mL |
2.1 × 108/mL |
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A, End-point assay was read at day three by scoring GFP-positive wells. B, End-point assay was read at day seven by scoring wells positive for CPE. Median tissue culture infectious dose.
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Long black and white boxes represent DNA sequences for the target and the reference genes. Dashed lines and arrows represent the newly synthesized chains and directions from PCR. When more copies of target sequences are available, set B primers bind more frequently to them than to the reference sequences, forming less PCR product from reference gene. Modified from Deng and Kim (1999 p788).
Discussion
BV infects non-invertebrates only. Autographa californica nuclear polyhedrosis virus is the most used virus with insect Spodoptera frugiperda sf9 and sf21. Rohmann (1988).Polyhedrosis virus has a two-phase life, early and late span on BV. It enters the insect midgut by endocytosis or by fusion. Viral DNA is uncoated at the nucleus integrating into the insect genome thus initiating replication.
The early phase form budding extra cellular virus particles, which in turn infect neighboring cells, thus generating occluded virus particles in the late stage. The occluded particles are embedded in matrix made of polyhedrosis protein. (Summer and smith 1978 p406). For 5-6 days, post infection the occluded virus continue to develop and can be detected by microscopy as dark polygonal bodies which usually fill the infected cell's nucleus. This shows presence of viral CPE on the cells. The polyhedron has an approximate molecular weight of 29kDa. (O'Reilly et al 1992).
Towards the end of late phase, polyhedron protein is synthesized at a high speed of up to 1mg/1-2 million infected cells.
However, the protein is not necessary in the BV lifecycle but is essential for transmission of viruses into the environment and other susceptible insect hosts. Polyhedrosis virus is co-infected to sf9 cells gene of interest and the polyhedron gene is replaced by recombination in the polyhedrosis virus genome thus regenerating a recombinant virus. The recombinant virus produces recombinant protein and other additional recombinant viruses. (Summers and smith 1987).
Detection of PCR gel image of the BV polyhedron gene is easily detected after amplification as shown in the results above. The presence of definitive amplicons show presence of BV, The Sf9 assay scored at 7 days post-transfection for CPE and gave comparable results to the 3-day GFP assay.