KR19990065232A - Fluorescent silkworm and manufacturing method using recombinant baculovirus with green fluorescent protein gene inserted - Google Patents

Abstract

The present invention is to produce a recombinant virus (Bacterial Accession No. KCTC0418BP) inserted into the green fluorescent protein gene under the control of the polyhedral protein gene promoter of insect baculovirus (AcNPV), silkworm body cavity injection The present invention relates to a fluorescent silkworm and a method for producing a recombinant silkworm, in which a green fluorescent protein gene is expressed by small-scale proliferation of a recombinant virus and regulation of a polyhedral protein gene promoter. The degree of expression of fluorescent silkworms differed according to the silkworm varieties. Especially, Daesunggum was effective in the expression rate and metastasis, and green fluorescence was emitted all over the body during ultraviolet irradiation. In addition, the green fluorescent protein gene of fluorescent silkworms was transferred to the genome by the mechanism of insertion into the genome of host insects. Therefore, the fluorescent silkworm development technology of the present invention can be utilized for the creation of transgenic insects with improved new function and genetic engineering.

Description

Fluorescent silkworm and manufacturing method using recombinant baculovirus inserted with green fluorescent protein gene

The present invention is a recombinant AcNPV (institute of biotechnology research institute strain KKTC 0418BP) incorporating a green fluorescent protein gene under the control of a polyhedral protein gene promoter of insect baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV or less AcNPV) It relates to a green fluorescent silkworm silkworm silkworm (Bombyx mori) and a method for producing the same.

Silkworm silk is a insect that produces silk, familiar with people through mass rearing in both East and West, and recently, it is also used for education and pets. Silkworms are larger in size than other insects, are easy to breed in large quantities, do not cause allergies in humans, and are well-studied by genetic breeding. Recently, the development of transgenic insects using silkworms or the mass production of recombinant proteins by baculovirus expression systems (Mori et al., Bio / Technology 13, 1005-1007, 1995), Schoudary et al. , Methods in Molecular Biology, Baculovirus Expression Protocols, ed Richardson, CD, 243-264, Human Press, Totowa, NJ, 1995), Maeda, Insect Cell Biotechnology, eds Maramorosch, K. Mclntosch, A., 1- 31, CRC Press, Boca Raton, Fl., 1994).

Recently, Chapel (Chalfle et al., Science, 263, 802-805, 1994) et al. Has developed a green fluorescent protein as a visual marker for the location and expression detection of proteins in living organisms, but only ultraviolet or 395-470 nm wavelengths. Irradiation of the intracellular green fluorescent protein is possible by irradiation, and since the substrate and the cofactor are not required, the use thereof is not limited. Insect baculovirus has been found to have host specificity by Granados Federichi, The biology of baculoviruses, Vol, l, ll, Boca raton, CRC Press.

More recently, Mori et al. (Bio / Technology, 13, 1005-1007, 1995) reported that AcNPV multiplies small amounts in silkworm larvae and uses it to control light-emitting genes in fireflies under the control of the Drosophila heat shock protein gene promoter. Injected recombinant AcNPV was injected into the silkworm and luminescence was also confirmed in the dead silkworm by injection of luciferin and chemical as a substrate.

Also, according to Hung et al., J. Invertebr. Pathol., 69, 234-245, 1997, etc., recombinant AcNPV produced by inserting a firefly luminescent gene under the control of AcNPV polymorphic protein gene was injected into silkworm larvae. In silkworm larvae, small amounts of luminescent genes were reported. However, the above reports have a problem that the silkworm eventually dies in order to confirm the expression of foreign genes in silkworms, and the method of identification also hassles due to the use of substrates and chemicals. In other words, their use and application are quite limited because they are not easily identified in the living state.

The present invention uses a recombinant insect baculovirus expression system to produce a recombinant AcNPV (Bacterial Cycle Accession Number KCTC 0418BP, hereinafter referred to as a recombinant virus) in which a green fluorescent protein gene is contained under the control of a polyhedral protein gene promoter. By injecting into other silkworms, the recombinant virus multiplies in small amounts without dying of the silkworms and expresses the green fluorescent protein by the regulation of the polyhedral protein gene promoter, and it is easily confirmed by ultraviolet irradiation and found that the gene is transferred to the next generation. Since the present invention is a technology capable of confirming the introduction and expression of foreign genes in a living state for the first time in the world, it is possible to create a transgenic insect improved in new function and genetic engineering using green fluorescent protein gene, and improved by the method of the present invention. Fluorescent silkworms emit beautiful green fluorescence during UV irradiation. The fluorescent silkworm manufacturing technology proposed a new model that enables the production of useful substances in living silkworms, as well as the creation of new functional and genetically engineered transgenic insects and animals, based on the technology of the present invention for exhibition and pet use. It can be used for special purpose insects and animals.

1 is a schematic diagram of the production of recombinant baculovirus containing a green fluorescent protein gene under the control of a polyhedral protein gene promoter.

FIG. 2 is fluorescent silkworms produced by the recombinant baculovirus of FIG. 1.

Figure 3 is a result of Western blot analysis of the body fluid protein synthesis of fluorescent silkworms and silkworms using protein electrophoresis and green fluorescent protein antibody.

Figure 4 compares the green fluorescent protein expressed in the fat tissue of the next generation fluorescent silkworm with the fat tissue of the common silkworm with a fluorescence microscope.

FIG. 5 shows the results of analysis of the transfer of the green fluorescent protein gene from the next-generation fluorescent silkworm genome by polymerase chain reaction (PCR).

In order to express green fluorescent protein genes in silkworm larvae by control of polyhedral protein genes of recombinant AcNPV having different host ranges, the production of recombinant AcNPV under the control of a gene promoter and the injection of them into silkworm larvae produce green fluorescent silkworms and the silkworm body. Expression of green fluorescent protein by polymorphic protein gene promoter regulation and easy identification by ultraviolet irradiation from live fluorescent silkworm and confirmation of next generation transfer of the gene and expression in next generation. The present invention is described in more detail by the following examples, which do not limit the scope of the present invention.

Example 1

(Production of Recombinant Virus)

To prepare a recombinant virus of AcNPV under the control of the green fluorescent protein gene under the polyhedral protein gene promoter, pGFP (manufactured by Clontech) and restriction enzymes BamHl and Pstl of the baculovirus transfer vector pBacPAK8 (manufactured by Clontech) as shown in FIG. A green fluorescent protein gene of about 720 bp cut from was inserted. 500 ng of recombinant transfer vector (pAc-GFP) and BacPAK6 virus DNA (Clontech) cut with Bsu361 were mixed, and the same amount of lipofectin (lipofectin, Gibco) was added and allowed to react at room temperature for 15 minutes. The mixture was inoculated into a 35 mm 2 cell culture dish containing 1.0 × 10 6 insect cell lines (Sf9), transfected at 27 ° C. for 5 hours, and incubated at 27 ° C. for 5 days after replacement with a new culture medium. .

Selection of recombinant virus (Ac-GFP) was done by plaque assay (Summers Smith, Texas Agriculure Experiment Station, Bulletin No. 1555, 1987). The selected recombinant virus was propagated in Sf9 insect cell line.

The schematic diagram of the recombinant baculovirus is shown in FIG. 1, and the green fluorescent protein gene was inserted under the control of the polyhedral protein gene promoter of AcNPV.

Example 2

(Production of Green Fluorescent Silkworm by Recombinant AcNPV)

For the production of green fluorescent silkworms, the recombinant virus was purified by ultra-centrifugation (23,000 rpm, 1 hour, 4 ° C) and dissolved in sterile distilled water. The recombinant virus was injected into the body by about 2.5 × 10 4 PFU per silkworm on day 1 of 5 years of age, which was stunned slightly on ice. About 20 varieties of silkworms were used, including Daesung-jam (hybrids of Japanese and Chinese) and the nap system. Silkworms waking up immediately after injection were kept at 25 ° C. with mulberry leaves.

Fluorescent silkworm was produced by irradiating ultraviolet rays at a predetermined time every day, and the results are shown in FIG. 2. Common silkworm (a) did not emit green fluorescence even after irradiation with ultraviolet rays, whereas Daesung jam began to fluoresce green from the breast paws after 4 days of recombinant virus injection and showed green fluorescence from the entire body on day 7 (b ). In addition, silkworm varieties of the nap system showed beautiful fluorescence due to the combination of colored patterns and green fluorescence (c). When ultraviolet rays were irradiated to Daesung sleep fluorescent silkworm on the mulberry branch, green fluorescence was emitted from the whole body in the living state (d).

Example 3

(Blood Protein Synthesis of Fluorescent Silkworms and Common Silkworms)

In order to compare the synthesis of humoral protein synthesis between fluorescent silkworms and normal silkworms by injecting recombinant viruses, body fluids were collected at daily intervals, followed by protein electrophoresis (Laemmli, 227 680-685, 1970) and western blot. The analysis was performed. Two times of protein sample solution (0.0625M Tris-HCL, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.001% bromophenol blue) was mixed with 1ul of body fluid collected at each time. After heating at 100 ° C. for 5 minutes, electrophoresis was performed on 12.5% SDS-polyacrylamide gel and stained with Coomassie brilliant blue.

In addition, Western blot analysis was performed by transferring the protein samples of the gel not stained with Coomassie Brilliant Blue to the Nitrocellulose membrane and binding the antibody against the green fluorescent protein. The results are shown in FIG. 3.

Electrophoresis of blood protein synthesis patterns of common silkworm (lane 1) and fluorescent silkworm (lane 2) taken 7 days after injection showed that the band pattern was indistinguishable. However, Western blot analysis showed no visible silkworm (lane 3), whereas fluorescent silkworm (lane 4) was able to clearly identify a band of about 27 kDa ultra-fluorescent protein present in the blood on day 7. At this time, the growth rate of recombinant AcNPV virus present in the fluoridal silkworm body fluid was increased by about 240 times.

Example 4

(Next-generation transition to fluorescent silkworm)

Next-generation transition of fluorescent silkworms was confirmed by UV irradiation of the next generation 5 silkworm larvae, and the next-generation transition rate was relatively low 10.9%. In addition, the fat tissue of the fluorescent silkworm was extracted and CKF was irradiated with a fluorescence microscope, and the results are shown in FIG. 4. In the case of fluorescent silkworms (a and b in Fig. 4) and ordinary silkworms (c and d in Fig. 4), green fluorescence is shown in the fat body of fluorescent silkworm in Fig. 4b, which is green fluorescence. It can be seen that the gene of silkworm is transferred to the next generation.

In addition, genomic genes were extracted and analyzed by PCR (Polymerase chain reaction) using specific primers to analyze gene transfer in next generation fluorescent silkworms.

Genomic gene extraction was performed at 37 ° C. for at least 4 hours by adding proteinase K to a concentration of 0.5 mg / ml (manufactured by Sigma) to fat tissue. Then, the same amount of phenol / chloroform-isoamyl alcohol (phenol / chloroform-isoamylalcohol, 24: 1) was added and mixed, and the supernatant was collected by centrifugation at 15,000 rpm for 5 minutes. Two times cold ethanol was added thereto, centrifuged at 15,000 rpm for 15 minutes to precipitate DNA, and the recombinant viral DNA separated by 70% ethanol washing was dissolved in sterile distilled water and used for PCR analysis.

PCR primers used two primers, 5'-ATGAGTAAAGGAGAAGAA-3 'and 5'-TTTGTATAGTTCATCCAT-3' at the termination site of the green fluorescent gene to analyze the presence of the green fluorescent gene, and also the green fluorescent gene AcNPV. Two primers, 5'-CTGATATCATGGAGATAA-3 'and 5'-TTTGTATAGTTCATCCAT-3' of the AcNPV polyhedral protein promoter region, and 5'-TTTGTATAGTTCATCCAT-3 'of the green fluorescent protein gene termination region, were used to determine whether the polymorphic protein gene promoter region of Was used.

PCR reactions resulted in 50ul of 250ng genomic DNA, 2.5 U Tak DNA polymerase (promega), 200nM deoxynucleotide triphosphate, 100pM of each primer and 3mM MgCl2. And PCR (Thermal Cycler, Perkin Elmer Cutus). The reaction temperature and time were reacted for 1 cycle at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C for 35 cycles. After PCR reaction, the product was precipitated in cold ethanol and analyzed on 1.0% agarose gel.

As shown in FIG. 5, PCR products were not detected in the genome of the common silkworm of lane 3, whereas the fluorescent silkworm contained the AcNPV polyhedral protein gene promoter region as well as the green fluorescent protein gene (lane 1). (Lane 2), and no recombinant virus was detected in the blood of the next-generation fluorescent silkworm. Therefore, it was confirmed that the transfer of fluorescent silkworms is a next generation transfer of the green fluorescent protein gene into the mechanism of insertion into the genome of the host insect.

The present invention is a technology capable of confirming the introduction and expression of foreign genes in a live state, using the green fluorescent protein gene can create a new functional and genetically modified transgenic insects, the fluorescent silkworm improved by the method of the present invention Fluorescent silkworm manufacturing technology, which emits beautiful green fluorescence when irradiated with UV light, presents a new model that enables the creation of new functional and genetically engineered transgenic insects and animals as well as the production of useful substances in living silkworms. It can be used for special purpose insects and animals.

Claims (6)

Recombinant baculovirus in which green fluorescent silkworms are inserted with the green fluorescent protein gene under the control of the polyhedral protein gene promoter of insect baculovirus (AcNPV) (KCTC 0418BP, Korea Institute of Science and Technology). The method of claim 1, Recombinant baculovirus with green fluorescent protein gene characterized in that silkworm larvae contain recombinant virus (KCTC 0418BP) with green fluorescent protein gene inserted under the control of the polyhedral protein gene promoter of insect baculovirus (AcNPV). Green fluorescent silkworm using. The method according to claim 1 and 2, Green fluorescent silkworm transformed by recombinant baculovirus (KCTC 0418BP) inserted with green fluorescent protein gene. Insert a 720 bp green fluorescent protein gene cut from pGFP into the restriction enzymes BamHl and Pstl of the baculovirus transfer vector pBacPAK8, mix the BacPAK6 virus DNA cut with 500 ng of recombinant transfer vector (pAc-GFP) and Bsu361) Pectin is added and reacted to inoculate insect cell line (Sf9) to transfect, culture and select the recombinant virus to grow in the Sf9 insect cell line, and the recombinant baculovirus gene is inserted under the control of the polyhedral protein gene promoter of AcNPV. Method for producing a green fluorescent silkworm using recombinant baculovirus (KCTC 0418BP) in which the green fluorescent protein gene is inserted. The method of claim 4, wherein Recombinant baculovirus into which a green fluorescent protein gene is inserted is characterized in that the recombinant virus is purely separated by an ultrafast centrifuge, dissolved in sterile distilled water, stunned on ice, and injected into the silkworm larva to express the foreign gene in the body. KCTC 0418BP) manufacturing method of green fluorescent silkworm. The method according to claim 1, wherein Under the control of the polyhedral protein gene promoter of the insect baculovirus (AcNPV), the baculovirus (KCTC) into which the green fluorescent protein gene is inserted is expressed by containing a recombinant virus containing the green fluorescent protein gene in an animal or an insect. 0418BP).