Baculovirus is a larger type of insect virus. It can not only be used as an important biological insecticide, but also can be used in baculovirus vector expression system. It has excellent performance in agriculture, basic molecular biology and medicine. Its important role. In the field of biological science research, the use of a baculovirus vector expression system to express recombinant proteins has the characteristics of high safety, ability to accommodate larger foreign genes, and easy screening of recombinant viruses. Common types of baculovirus expression vector systems include Bac-to-Bac system and baculovirus-S2 system.
Baculoviruses are a kind of pathogenic microorganisms that specialize in parasitizing arthropods. They are named after their virus particles have a rod-shaped structure. The baculovirus family includes two species of nuclear polyhedrosis virus and granulovirus. At present, only nuclear polyhedrosis virus is used as a foreign gene expression vector, and the most widely used is Autographa californica nuclear polyhedrosis virus (ACMNPV) under the nuclear polyhedrosis virus. Baculoviruses use insects as hosts. Infected insects are usually acute infections and cause a large number of deaths of infected insects. When baculovirus is used in the construction of expression vectors, one or more baculovirus promoters can be used, and after the foreign target gene is inserted into the promoter, a recombinant virus can be obtained. This recombinant virus replicates in insect cells or in the insect body while allowing foreign genes to be expressed. The baculovirus vector expression system has perfect post-translational processing and modification and the ability to efficiently express foreign genes. The main types of common baculovirus vector systems are as follows:
The Bac-to-Bac expression system is a transposon-mediated baculovirus recombination system. Some researchers have used the Bac-to-Bac baculovirus expression system to express mouse soluble IL-5α receptor (sIL-5Rα) protein , And to identify its antigenicity [1]. The method is to use Vector NTI Advance software to optimize the extracellular region of mouse IL-5Rα gene to synthesize the sequence, clone sIL-5Rα into pFastbacI, construct the recombinant shuttle plasmid pFastbacI-sIL-5Rα, transform competent E. coli DH10BacTM, construct Recombinant bacmid Bacmid-sIL-5Rα was transfected into sf9 cells, and baculovirus was obtained by heterologous gene recombination. The virus titer after amplification was detected by the Reed-Muench method; P3 and P4 generation viruses were infected with sf9 cells at 0.05 MOI. The expression of sIL-5Rα protein was identified by SDS-PAGE, the expression product was purified by cation exchange chromatography, and the purified sIL-5Rα protein was identified by SDS-PAGE and Western blot. Medicilon researchers have established a mature baculovirus-insect cell expression system service platform, providing services including recombinant baculovirus preparation, expression and purification of recombinant proteins and their complexes.
The researchers found that the recombinant bacmid Bacmid-sIL-5Rα was identified by PCR, which proved that the construction was correct; the virus titer of the P3 generation recombinant baculovirus was 1.7×107pfu/ml; the expressed and purified sIL-5Rα protein had a relative molecular weight of approximately Specific protein bands can be seen in 43 000 places, and the lysed supernatant of infected sf9 cells can specifically react with the rabbit anti-mouse IL-5Rα polyclonal antibody. Therefore, the Bac-to-Bac baculovirus expression system can successfully express the mouse soluble IL-5Rα receptor protein, which lays an experimental foundation for further research on its biological functions and therapeutic effects in asthma model animals.
Researchers also used the Bac-to-Bac baculovirus expression system to express mouse glucocorticoid-induced tumor necrosis factor receptor ligand (G ITRL) protein. Results The Bac-to-Bac baculovirus expression system was used to successfully express the mouse GITRL protein, which laid the experimental foundation for further study of its biological activity and function.
The baculovirus-S2 system uses recombinant baculovirus to transfect Drosophila S2 cells. In the baculovirus-S2 expression system, the Drosophila promoter replaces the baculovirus polyhedrin gene promoter, including heat shock. Promoters such as protein 70 gene, actin 5c gene and metallothionein gene. Through positive regulation, they can each drive high-level expression of downstream target genes. Due to the application of strong promoters and inducible expression factors, almost all cells infected with recombinant baculovirus will show signs of expression. The target protein expressed by the baculovirus-S2 system can reach about 20% of the total cell protein, and its expression level is much higher than other eukaryotic vector systems, so it is a very promising baculovirus expression system.
At present, the baculovirus has been developed as a gene expression vector and applied to the baculovirus vector expression system of insect cells. It has been widely used in the expression research of foreign target genes. The use of baculovirus expression vectors has the advantages of large gene capacity, good operability, high expression efficiency, good biological safety, and strong targeting. It will be more and more widely used in the field of medical research and development, and its development prospects will also be more and more. it is good.
[1] Expression and identification of mouse soluble IL-5α receptor in Bac-to-Bac system [J].
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