Baculovirus Expression Vector System and its Application

The most researched insect baculovirus is Autographa californica multiple nuclear polyhedrosisvirus (AcMNPV), its host is Spodoptera frugiperda (Sf); Bombyx mori nuclear polyhedrosis virus (Bombyx) mori nuclear polyhedrosis virus (BmNPV), whose host is the silkworm (Bombyx mori), these two baculovirus vectors are often used to express foreign proteins [2,3].
The gene expression of baculovirus is divided into four stages: early expression, early expression, late expression, and very late expression. Among them, there are two highly expressed proteins in the very late gene expression process: polyhedrin and P10 protein. The promoters of the two genes have strong promoters. The polyhedrin gene polh is a very late gene with extremely high expression. The accumulation in the cell during the later stage of infection can be as high as 30% to 50%. When the polh gene is deleted from the genome, its progeny viruses cannot form polyhedrons, but not It does not affect the replication of the virion. Therefore, the polh gene site is an ideal foreign gene insertion site for the baculovirus expression system [2]. P10 is also an extremely late gene that is highly expressed, and it is also a non-essential gene for virus replication. Therefore, the P10 site is often used as an insertion site for foreign genes.

2.1 The Advantages of the Baculovirus Expression Vector System

The baculovirus expression system has the following advantages [11,12,13]: (1) High safety: The host of baculovirus is limited to invertebrates, and it is not infectious to humans, animals and other vertebrates, and has strong specificity. Compared with adenovirus vector (Adenovirus vector) and lentivirus vector (Lentivector), it is safer. (2) High expression efficiency: The baculovirus very late polyhedrin promoter and P10 protein promoter are used to express foreign proteins strongly, and the expression level can reach up to 50% of the total protein content of the infected cells. The P10 protein is a non-essential segment in the viral genome, and the insertion of foreign genes does not affect the replication and infection of the virus. (3) The expressed protein has biological activity: Compared with the prokaryotic expression system, insect cells are eukaryotic cells. The way they modify and process the protein after expression is similar to that of mammalian cells, and can recognize and correctly carry out signal peptides. Removal, phosphorylation, glycosylation and other reactions, and make the exogenous protein fold correctly, disulfide bond matching and oligomer formation in the cell, making it close to the natural protein in structure and function. (4) When the foreign gene is inserted into the polyhedrin gene locus, it will inevitably cause the deletion or inactivation of the latter. Therefore, the recombinant virus will not be able to produce polyhedron. This not only provides a selection marker for the recombinant virus, but also the recombinant virus It cannot exist in the environment like wild viruses, and it is safer. (5) Large capacity: The baculovirus genome is about 130Kb, with multiple strong natural promoters. It is also easy to construct new artificial promoters, which can express multiple genes at the same time and can accommodate large fragments of foreign genes. (6) The recombinant virus constructed based on BmNPV has a narrower host range. Except for the silkworm, there are basically no other hosts, and the silkworm has no flight ability and is completely raised by humans. Moreover, the research on the silkworm has been going on for many years, and the genetic background is relatively clear. , The large-scale breeding technology is mature, and the cost is greatly reduced. It is precisely because of the advantages of this expression system that the genes of viruses, bacteria, fungi, animals and plants have been highly expressed in insect cells or larvae.

2.2 Construction Strategy of Baculovirus Expression Vector System

Because of the relatively large genome of baculovirus, the cloning of foreign genes cannot be inserted directly through restriction enzyme digestion and ligation, and must be mediated by a transfer vector. When constructing the transfer vector, clone the flanking sequences of the coding region of the genes to be inserted into the baculovirus site (such as the polyhedron gene and the P10 gene) into the same plasmid vector, and then clone the foreign gene and the corresponding promoter in the two Between segments of the flanking sequence. After the transfer vector is successfully constructed, the vector and wild-type baculovirus are co-transfected into insect cells, and homologous recombination occurs in the cell through the homologous border regions on both sides, and the foreign genes and promoters replace the corresponding regions of the baculovirus. Recombinant virus. If the position of the polyhedrin gene is inserted, the recombinant virus will not produce polyhedron, which can be used to distinguish the recombinant virus from the wild virus, and then the recombinant virus can be purified through multiple rounds of plaque experiments. However, due to the extremely low recombination efficiency of wild-type viruses, only 0.1% to 1%, and the phenotypic difference is not significant, the screening of recombinant viruses is difficult, and there are certain difficulties in application. Through continuous exploration in recent years, scholars have inserted some screening genes into the transfer vector, such as β-galactosidase, TK gene [14], Neo gene [15], or modified wild-type baculovirus, such as Cre- Loxp site-directed transposition[16,17], linearization, Bacmid, etc., not only have greatly improved the efficiency of baculovirus recombination, but also the screening and identification of recombinant baculovirus has become more and more convenient and easier.

2.2.1 Insert Β-Galactosidase Gene for Blue and White Screening

In the construction of plasmids, blue-white spot screening is an important method for recombinant screening. Bacteria containing the complete LacZ reading frame, under the action of the inducer IPTG (isopropyl-β-D-thiogalactoside), the chromogenic substrate X-Gal (5-bromo-4-chloro-3- Indole-β-D-galactoside) produces blue colonies when it is present, so it is easy to identify.
In 1990, Viaard et al. [18] used the P10 gene promoter to drive the LacZ gene in the upstream of the polyhedrin gene, and used the polyhedrin promoter to drive the foreign protein to construct the transfer vector pJVNhe I. After co-transfecting it with wild-type AcMNPV into Sf9 cells, the recombinant virus has no polyhedron production and can express β-galactosidase. By adding X-Gal to make it form blue plaques, it can be easily recombined Virus screening. Although this method is convenient for recombinant virus screening, the virus recombination efficiency is still very low, with a recombination rate of only about 0.1%-1%.

2.2.2 Linearization of Baculovirus

Kitts et al. [19] introduced a single endonucleation site (Bsu36 I) into the baculovirus polyhedrin gene locus based on the feature that linear DNA is more prone to recombination than general circular DNA. Previously, after cutting the baculovirus DNA with Bsu36I, the covalently closed circular dsDNA was linearized. After transfecting the cells, about 30% of the progeny viruses were recombinant viruses.
In 1993, Kitts and Possee improved this system and combined it with visual selection and copy selection [20]. Add the LacZ gene to the polyhedrin locus of the baculovirus, and introduce a Bsu36 I site on each side of the polyhedrin gene (in ORF1629 and ORF603 genes). This modified AcMNPV is called the BacPAK6 mutant. After Bsu36 I digestion, it not only linearized the baculovirus DNA, but also removed the two genome fragments mentioned above, destroying the open reading frame ORF1629. Because ORF1629 is necessary for baculovirus replication, after the loss of these two fragments, only after recombination with the transfer vector plasmid DNA, ORF1629 can be restored to produce an active virus. At the same time, the recombinant virus does not have LacZ. When IPTG and IPTG are present, they will appear white, which is more helpful for the screening of recombinant viruses. The frequency of this method to produce recombinant virus is as high as 85%-99%.

2.2.3 Bac-To-Bac Baculovirus Expression Vector System

In 1993, Luckow and other researchers successfully developed a rapid and efficient technology for producing recombinant Autographa californica nuclear polyhedrosis virus (AcNPV), which uses the principle of bacterial Tn7 transposon to complete the recombinant virus in E. coli Construction, named Bac-to-Bac expression system [21,22,23], which means from bacteria (bacterium) to baculovirus (baculovirus), revolutionary changes in the construction method of recombinant insect baculovirus (Figure 1.1).
The system uses bacterial mini-F replicon and Tn7 transposon to construct a baculovirus shuttle vector Bacmid, which contains bacterial single copy number mini-F replicon and Kanamycin (Kanamycin, Kan+) resistance selection marker Gene and a partial DNA fragment encoding β-galactosidase α peptide, and introduced the bacterial att Tn7 transposon target site. The baculovirus shuttle vector can grow in Escherichia coli like a plasmid and is infectious to lepidopteran insect cells. It (130kb) was transformed into Escherichia coli (E.coil) DH10, and the transformant was obtained and named DH10 Bac. There is also a helper plasmid (13.2kb) in DH10, which encodes Tn7 transposase and contains Tetracycline (Tet+) resistance gene. There is a complete expression cassette between the left and right arms of the mini-Tn7 transposon on the donor plasmid pFastBac, including the gentamicin (Gentamincin, Gen+) resistance gene, the baculovirus PPH promoter (or PP10 promoter), Multiple cloning sites and SV40 polyA and other elements. The foreign gene is inserted into the multiple cloning site downstream of the baculovirus promoter, and the recombinant donor plasmid is transformed into DH10Bac competent cells. Under the action of the encoded transposase, the donor plasmid is transformed by the mini-Tn7 transposon The upper expression cassette is inserted into the target site of Bacmid, disrupting the expression of lacZ α gene. Screening on a culture plate containing Kan+, Gen+, Tet+, IPTG and X-Gal, the colonies of recombinant Bacmid (recombinant viral genome) transformants will be white, while the colonies of non-recombinant Bacmid transformants will still be blue. Therefore, the recombinant virus can be screened by the color of the colony. By selecting a single white colony for culture, extracting the recombinant Bacmid genome, and then transfecting it into insect culture cells to obtain a recombinant virus, the recombinant protein can be expressed and produced.
The Bac-to-Bac system based on BmNPV was successfully established by Motohashi et al. in 2005 [24]. Wu Xiaofeng and Cao Cuiping et al. modified the BmNPV viral genome [25,26], inserting a bacterial strain at the polyhedron site. Copy the mini-F replicon, a partial DNA fragment encoding β-galactosidase α peptide (ie lacZα gene) and a fragment of the kanamycin resistance gene to complete the silkworm BmBacmid. Then Bacmid was transformed into E. coli DH10α, and the resulting transformant was named BmDH10Bac.

2.3 Application of Baculovirus Expression System

At present, a variety of genes have been expressed in recombinant baculovirus [2,3,27,28,29,30,], including viral genes, bacterial genes, plant genes, invertebrate and vertebrate genes, And human genes. The expression of these genes has played a great role in studying their functions, applications, prevention and treatment of diseases.

2.3.1 Recombinant Baculovirus used in Gene Therapy

Studies have found that although baculovirus is an insect-specific virus, it can enter mammalian cells and cannot replicate. Under the control of mammalian virus promoters (such as CMV immediate early promoter), foreign genes can be expressed in a variety of mammalian cells and are non-toxic to cells [31,32,33,34].
In 2003, Lindsay et al. proposed the idea of using baculovirus as a gene therapy vector to treat human prostate cancer [35]. In 2006, Liu Xiaoyun [36] et al. used the Bac-to-Bac system to construct a recombinant baculovirus Ac-CMV-Sox9, (Sox9 is a recognized therapeutic gene that can delay and reverse intervertebral disc degeneration), which proved that the Sox9 gene (Ac- CMV-Sox9) can be effectively expressed in rabbit nucleus pulposus cells cultured in vitro, and can promote the increase of type Ⅱ collagen content in the degenerated rabbit nucleus pulposus tissue cells to delay and reverse the degeneration of the intervertebral disc.
Because the baculovirus promoter is silent in mammalian cells, it cannot replicate and express in mammalian cells; at the same time, the baculovirus DNA is free in the cell and does not interact with the genetic material in the host cell and the body. The potentially defective virus integrates, so there is no risk of insertion mutation; baculovirus does not cause immune rejection in the body; it can be seen that BEVS can be used as a gene therapy vector with great development and application prospects.

2.3.2 Research on Genetic Engineering Vaccine Using Baculovirus as Carrier

As a carrier for genetic engineering vaccine research, baculovirus can be used to express subunit vaccine antigens, or it can be directly used as a DNA vaccine to be injected into the body to produce immune effects.
Wang SP[37] and others constructed a recombinant pseudotyped baculovirus vaccine modified by Porcine respiratory and reproductive syndrome virus (PRRSV) modified by ORF5 and ORF6 double gene co-expression. The mouse immunization test confirmed that the recombinant The levels of IFN-γ and neutralizing antibodies induced by the virus were significantly higher than those in the conventional DNA vaccine immunization group. Yu Guangqing [38] also used this system to construct a recombinant DNA vaccine expressing the glutathione S-transferase (GST) of Schistosoma japonicum, which also obtained better immune protection.
Xiaolin Zhang [39] et al. used the Bac-to-Bac system to construct recombinant BmNPV in 2009 and expressed Helicobacter pylori (Helicobacter pylori, Hp) heat shock protein A subunit (HspA) and urease B in silkworm pupae. Subunit (UreB), in the immune test of mice, found that Helicobacter pylori HspA and UreB expressed by oral silkworm pupa can produce specific immune responses in mice, and have immune protection and immunotherapy effects against Helicobacter pylori infection.
In addition, recombinant baculoviruses expressing pseudorabies virus glycoprotein [40], influenza virus hemagglutinin [41,42], rabbit hemorrhagic disease virus VP60 protein [43] and other DNA vaccines have also obtained good results The immune effect of the recombinant baculovirus DNA vaccine is a vaccine vector with good development prospects.

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