Purification experiments of recombinant baculovirus

Summary

The baculoviruses are a class of DNA viruses that exclusively infect arthropods in nature, and can be used (1) as genetically engineered viral insecticides to improve the efficiency of pest control (2) as ultra-efficient eukaryotic gene expression vectors for the production of useful engineered proteins (3) to study the structure and function of the baculovirus genomes (4) to study the mechanism of the regulation of eukaryotic gene expression.

Operation method

Encodes beta-galactosidase

Principle

Due to the large size of the insect baculovirus circular double-stranded DNA genome (about 130kbp) and the multiple recognition sites for restriction endonucleases, it is not possible to directly introduce exogenous genes into the viral genome by conventional genetic engineering operations. Insect recombinant baculoviruses are usually obtained in two steps. In the first step, the exogenous gene is cloned into a universal vector plasmid so that the exogenous gene is under the control of a strong baculovirus promoter and is flanked by baculovirus DNA sequences. These flanking sequences are not essential for viral replication, but are useful in the second step of homologous recombination. In the second step, this recombinant plasmid DNA and wild-type viral genomic DNA are introduced into insect cells, where the exogenous gene is inserted into the viral genome by homologous recombination (the exogenous gene replaces the wild-type viral genomic gene, such as the polycomb protein gene, which is located in the middle of the two flanking sequences), and the target recombinant viruses are screened out by a specific screening method and screening markers.

Materials and Instruments

baculovirus
Agar Bovine serum
Culture flasks Freezing tubes

Move

1. Prepare serial dilutions of viral supernatant obtained from cells transfected with pAC360 β-gal DNA in serum-free complete culture medium. Viruses form etiolated spots under an agarose top cover containing 150 ug/ml X-gal.2. When an etch has formed, pick a well-separated blue etch with a sterilized Pasteur pipette and transfer the agarose block into a sterile test tube containing 1 ml of serum-free complete culture medium. A series of ^10-1, ^10-2 and ^10-3 dilutions were prepared from the serum-free complete culture medium by vigorous hedging on a vortex mixer.3. Determine the virus by etching with an agarose top cover containing 150 ug/ml X-gal (helps to find recombinants) and repeat the etching test until a pure reservoir of recombinant virus is obtained.4. Pick a pure recombinant etch with a sterile pipette and transfer the agarose block into a 25 ^cm2 culture flask containing 2. ^5x106 Sf9 cells and 5 ml complete culture medium (containing 10% fetal bovine serum). Incubate at 27°C for 4-5 days until most of the cells are infected.5. Determine the viral titer (should be between ^5x107~1x108 pfu/ml).6. Add 1 ml of this virus reservoir to a 1.5 ml screw-top freezing tube for long-term storage at -80°C and store the remaining liquid (labeled as primary virus reservoir) at 4°C. Produce a large reservoir of virus from the primary stock and titrate it.

Caveat

Construct exogenous gene stable expression system insect cells due to the large size, irregular shape, easy to agglomerate, coupled with the virus infection after the cell expansion of about 1.5 times, these factors increase the difficulty of large-scale insect cell culture. At present, large-scale insect cell culture has been established in the form of (1) wall culture: wall culture can be divided into two kinds of rolling flask culture and microcarrier culture (2) suspension culture: suspension culture can be divided into shaking flasks and rotating flasks (3) immobilization culture: immobilization culture is a recent development of a new technology of large-scale cultivation of insect cells, insect cells, has two major advantages: it can effectively reduce the damage to the cells of the shear, at the same time, the cells and the cultivation of the culture culture of the cells, the cell and the cell culture of the cell culture. Insect cells have two advantages: it can effectively reduce the damage of shear force on the cells, and at the same time, the cells can be effectively separated from the training liquid, simplifying the operation steps and reducing the chance of bacterial infection. It can be divided into microencapsulation culture and stacked-bed culture, in which the stacked-bed culture combines the advantages of various culture methods, the insect cells grow on the wall on the spherical particles, and in the air-lift reactor in the descending area to form a stacked bed, while the ascending area for deep aeration and oxygenation. The packed-bed method provides a high culture surface and at the same time ensures adequate oxygenation.

Common Problems

1. Rod-shaped virus is a class of DNA viruses that exclusively infect arthropods in nature, the virus particles are in the form of rods, the genome is a double-stranded circular DNA molecule, and the DNA is compressed and packaged in the form of superhelix inside the rod-shaped capsid, with the size of 90-180 Kb. At present, rod-shaped viruses are widely used in pest control as highly efficient and safe pollution-free biological insecticides.

2. E. coli-insect cell shuttle vector screening system[10] The E. coli-insect cell shuttle vector (bacmid) is by far the most convenient and fastest recombinant baculovirus screening system. bacmid can replicate in E. coli like a plasmid and infect insect cells like a virus. bacmid is a recombinant baculovirus containing a mini-F replicon, a kanamycin replicon, a baculovirus, a baculovirus, a baculovirus and a bacmid. -Bacmid is a recombinant virus containing a miniF replicon, a kanamycin resistance screening marker, and the target sequences attTn7e and LacZ fragments of the bacterial transposon Tn 7. The role of the transfer vector is hereby replaced by the contributing plasmid. The contributing plasmid was designed to contain the gentamicin-resistant gene and the polycomb protein promoter-controlled exogenous gene flanked by the left and right arms of tn7, respectively. At the same time, in order to generate site-specific transposition, a helper plasmid (which is a marker for tetracycline resistance screening) is required to provide the transposon protein. Therefore, when the bacmid, the contributing plasmid, and the helper plasmid are transformed into the same strain, the transposon protein produced by the helper plasmid transposes the exogenous genes controlled by the gentamicin-resistance gene and the polycomb protein promoter to the attTn7 sequence of the bacmid, and at the same time, destroys the reading frame of the Lac-Z. This way, as long as the strain transformed with the above mentioned plasmids is transformed in a strain containing kanamycin, tetracycline, and X-gal, it is not necessary for the strain to be transformed into a strain containing kanamycin, tetracycline, and X-gal, Thus, by screening the transformed plasmid on LB medium containing kanamycin, tetracycline, gentamicin, X-gal and IPTG, the composite Bacmid can be obtained, and the composite Bac midDNA is purified and transfected into the insect cells, the pure recombinant virus is obtained, and the exogenous genes are expressed under the control of the polycomb promoter in the insect cells. This system is the most convenient and fastest system in BEVS, and the whole cycle of recombinant virus screening can be shortened to 7-10d, and can be continuously screened, so it is very suitable for the construction of cDNA library and protein engineering research applications. At the same time, the contributing plasmid is smaller than the conventional recombinant transfer vector, which can construct more restriction sites and alteration to facilitate the insertion of exogenous genes, and the contributing vector is not only suitable for BEVS, but also suitable for transposition in other eukaryotic expression systems to express exogenous genes. Due to the immunity of transposition, multiple insertion of exogenous genes can be prevented. The disadvantage is that only 12.5kb of the exogenous gene is inserted into the polycomb protein locus, and the insertion may destroy the working environment of the polycomb promoter, resulting in a slight decrease in the expression of the exogenous gene compared to the traditional homologous recombinant purified recombinant virus.

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