KR100325394B1 - Novel transfer vector for eukaryotic cell, recombinant expression virus vector and process for preparing recombinant protein using the same - Google Patents

Abstract

The present invention provides a gene cloning delivery carrier for eukaryotic cells prepared using genomic DNA of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), a recombinant virus for expressing a foreign gene cloned in the delivery carrier, and the recombinant virus. Polyhedrin gene and its promoter present in 7300 base-pair DNA fragments produced by digestion of the genome DNA of the white fire moth polyhedron virus (HcNPV) isolated from Korea with EcoR I restriction enzyme. Eukaryotic gene cloning delivery carrier (vector) pHcEV-IV was prepared using the recombinant virus lacZ-HcNPV (Accession No. KFCC 11109) to express the foreign gene cloned in this delivery carrier and then to the recombinant virus. The inserted lacZ gene was transferred to the insect cell Spodoptera frugiperda ce. ll line: IPLB-SF21) has an excellent effect on mass production of beta-galactosidase enzyme.

Description

Novel transfer vector for eukaryotic cell, recombinant expression virus vector and process for preparing recombinant protein using the same

The present invention relates to a novel recombinant gene delivery vehicle for eukaryotic cells and foreign gene expression recombinant virus carrier and a method for producing a recombinant protein using them. More specifically, a novel recombinant gene delivery vehicle for eukaryotic cells and exogenous gene expression recombinant virus carriers are prepared using the HphanNPa genomic DNA (HcNPV) genomic DNA. It is about.

Chromosomes in living organisms contain genes that hold information that makes proteins. Most genes are made of nucleic acid called DNA. The genetic material of eukaryotes and the genetic material of some DNA viruses consists of a double stranded chain, which consists of four bases: adenine (A), guanine (G), cytosine (C) and thymine (T). In composition. Double-stranded DNA is helical, with genes lined up in a straight line. Genetic information of genes is transcribed to make messenger RNA (mRNA), which is detoxified in the cytoplasm to produce proteins like ribosomes. The messenger RNA receives a transcription from a gene that produces a protein (recognized as three base pairs) from a gene (a DNA sequence that has a code for the sequence of amino acids in a single protein and is involved in synthesis) and connects the amino acids of the protein according to the code. To make the finished protein molecule. This process is called the central theory of molecular biology. Genetic engineering or recombinant genetic engineering techniques have been developed using these gene expression processes. Gene DNA can be expressed in cells of different organisms. The basic organism used in genetic recombination technology is bacteria. Because they are unicellular organisms, they are easy to handle and prefer to use bacteria. In addition, there is a genetic material made of cyclic double-stranded DNA called plasmid in bacterial cells. The plasmid is manipulated by recombinant DNA technology to transfer and express foreign genes (genes to be inserted into a carrier) in bacterial cells to make proteins. The basic factors necessary for genetic recombination or cloning (inserting a foreign gene with a linker) are firstly the foreign gene to be cloned, and secondly the restriction enzyme to cut the foreign gene. enzyme is an enzyme present in bacteria that is separated and purified, and this enzyme has the function of recognizing and cleaving a specific nucleotide sequence of DNA). There must be a delivery and expression carrier (vector is English name) that can be transported and expressed in the cell, and the fourth must be a host cell that will safely maintain and express the cloned recombinant gene. Among them, the function and composition of the expression and delivery vehicle have various functions depending on the recombinant species. Initially, cloning delivery and expression vehicles that could utilize E. coli were almost all. Since then, technology has evolved to develop a variety of delivery and expression vehicles that utilize a variety of bacteria.

Currently, in addition to the carrier using E. coli, a lot of achievements have been made in the development of carriers for eukaryotic cells. Among the eukaryotic cells, researches on the use of insect cells and nuclear polyhedron viruses, which are insect viruses, have been actively conducted, and carriers and insect cells using them have been developed. The expression of foreign genes in insect cells has several effects that cannot be obtained in prokaryotic cells. In other words, the protein to be expressed is modified to produce an active protein (O'Reilly et al., 1994, Baculovirus expression vectors, Oxford University Press Inc., New York). Development of the delivery vehicle using baculovirus, the insect virus, was mainly studied using Autographa californica nuclear polyhedrosis virus (Smith et al., Mol. Cell. Biol. 3: 2156-2165, 1983; Miller et al., In Genetic Engineering in Eukaryotes, ed.P. Lurquin & A. Kleninhpfs, pp89-97, New York, Plenum, and others Bombyx mori nuclear polyhedrosis virus) (Maeda, In Invertebrate Cell System Applications, ed. J. Mitsuhashi, pp 167-182. Boca Raton, Fla; CRC Press, 1989) and Limantris dispar nuclear polyhedron virus (Yu, Podgwaite & Wood, 1992, J. The development of delivery vehicles using General Virology 73: 1509-1514 has been reported. Thus, there is a demand for development of gene delivery and expression systems by various delivery and expression carriers in various cell lines. The reason for this is that because the characteristics of the delivery vehicle and the expression virus are different according to the characteristics of the foreign gene, the development of various systems may diversify the system for producing more recombinant proteins by expressing more genes in the eukaryotic environment. Because it can.

Molecular genetic studies on autographa californica nuclear polyhedrosis virus (AcMNPV), a parasitic nuclear polyhedron virus, have been studied by many scientists. Lee & Miller (J. Virology, 27: 754-767, 1978) and Smith & Summers (Virology 89: 517-527, 1978) compared the restriction fragment profiles of the genetic variants of AcMNPV. Nuclear polyhedrin (polyhedrin) gene of the polyhedrin virus (nucleus-forming protein) gene research has been active compared to the other. In particular, Smith et al. (Mol. Cell. Biol., 3,2156-2165, 1983) is a polyhedrin gene of AcMNPV by deleting the inclusion body (crystals of polyhedrin protein produced by the polyhedrin gene in insect cells, Mutants that contain viruses and are multifaceted and cannot form a virus with this inclusion body are called polyhedral viruses). These facts showed that polyhedrin synthesis and inclusion body formation were not essential for the growth of the virus. Summers et al. (J. Virology, 34: 693-703, 1980) show that in the case of AcMNPV, the polyhedrin gene is located in the restriction enzyme EcoRI-I fragment (10,000 base pairs). Estimated. Miller (J. Virology 39: 973-976, 1981) states that nuclear polyhedron viruses are rod-shaped nucleocapsids (nucleocapsids are viral nucleic acids, ie chromosomes in which proteins are bought), and have the ability to act as carriers. The gene is reported to have the ability to accept and extend genes larger than the original genome size. Recently, eukaryotic cell expression carriers using a powerful promoter of the polyhedrin gene of AcMNPV (a site where RNA polymerase adheres to promote transcription initiation) have been developed (Smith et al., Mol. Cell. Biol. 3: 2156-2165, 1983), they report two characteristics compared to other animal virus delivery systems. The first is that recombinant protein production is possible because of sufficient foreign gene expression and insect cell culture system established by transcriptional regulation of strong polyhedrin gene promoter. Second, the recombinant protein produced by this transport system is functionally complete. It is. These advantages are widely used in the cloning and expression of pharmaceutical and biologically important genes. Therefore, the restriction enzyme EcoRI-I fragment DNA (10 kb) of AcMNPV containing the polyhedrin gene was linked to the pUC9 and pBR322 carriers, which are E. coli plasmid carriers, to form a delivery vehicle, and then the foreign gene was transferred to the polyhedrin The gene was inserted immediately after the promoter of the gene, mixed with wild-type AcMNPV DNA in vitro to induce recombination, and co-transfection was performed on insect host cells to produce a eukaryotic cell delivery vehicle or expressing virus.

Recently, the isolated white-fly moth polyhedron polyhedrosis virus (HcNPV) has been successfully propagated in the insect cell, Spododotera luciferda cell line (LH, 1: 3-6, 1987). ). Lee & Lee (J. General Virology, 69: 1299-1306, 1988) isolated thermosensitive mutants of HcNPV and studied some of them. Lee et al. (J. Kor. Soc. Virology 20: 145-152, 1989) produced a restriction map of the HcNPV genome using restriction enzymes BamH I and Sma I. Lee et al. (J. Kor. Soc. Virology 21: 25-34, 1990) studied the cleavage of EcoR I, HindIII , BamH I and Sma I against HcNPV genomic DNA, and the α-32P-labeled AcMNPV polyhedrin gene. The fragment probe was used to confirm that the position of the polyhedrin gene of HcNPV was located in the EcoRI-H (7300 base pair) fragment, and Lee et al. (Molecules & Cells 2: 303-308, 1992) found that the base of the HcNPV polyhedrin gene The sequence was determined, and Park et al. (J. Kor. Soc. Virology 23: 141-151, 1993) prepared a primary HcNPV expression carrier having one Nco I insertion sequence using EcoRI-H (7.3 kb). Was named the pHcEV carrier.

As the use of genetic recombination technology has increased and diversified, the demand for eukaryotic cloning carriers and expression carriers has increased, and various developments are required. Therefore, the present inventors have studied in terms of protection of domestic industrial property rights. In order to meet the above requirements, the present inventors modified the pHcEV based carrier to make a highly functional delivery carrier for eukaryotic cells, and further, a recombinant virus carrier for expressing a foreign gene in an insect cell line, S. dopodera pruziferda cells. The beta-galactosidase gene of Escherichia coli was used as a search gene to identify cloning in the expression virus carrier, and a eukaryotic cell-expressing carrier system using a new genome was developed.

Accordingly, an object of the present invention is to provide a gene cloning delivery vehicle for eukaryotic cells prepared using genomic DNA of white bull moth polyhedron virus (HcNPV). Another object of the present invention is to provide a recombinant virus that will express the foreign gene cloned in the delivery vehicle. Another object of the present invention is to co-transfect the gene cloning delivery carrier for eukaryotic cells into which an exogenous gene is inserted into a spytopera pruperperda cell, which is an insect cell, with a white bull moth polyhedron virus to express a foreign gene. The present invention provides a method for producing the recombinant virus and a foreign gene product using the recombinant virus.

The object of the present invention is to eukaryotic by using a polyhedrin gene present in the 7300 base-pair DNA fragment generated by cleaving genomic DNA of DNA (HcNPV) isolated from Korea with EcoR I restriction enzyme. Gene Cloning for Cells The vector pHcEV-IV was prepared, and the recombinant lacZ-HcNPV (Accession No. KFCC 11109) to express the foreign gene cloned into this delivery carrier was prepared and the lacZ gene inserted into this recombinant virus. The expression of was achieved by confirming in the insect cells, Spodotera luciferda cells.

Hereinafter, the configuration and operation of the present invention.

FIG. 1 is a schematic diagram illustrating a manufacturing process of a pHcEV, a cloning delivery vehicle, using genomic DNA of Hyphantria cunea nuclear polyhedrosis virus (HcNPV).

Figure 2 is a schematic diagram showing the manufacturing process of pHcEVI, pHcEVII, pHcEVIII and pHcEV-IV by modifying the pHcEV.

Figure 3 shows the determined nucleotide sequence of the restriction site of the 13 cloning sites in the transporter pHcEV-IV.

Figure 4 is a schematic diagram showing the production of pHcEV-IV-lacZ recombinant plasmid by cloning the beta-galactosidase gene (lacZ) of Escherichia coli in the pHcEV-IV carrier.

Figure 5 is a recombinant virus lacZ which cotransfects the delivery carrier pHcEV-IV-lacZ DNA and the normal HcNPV HL-2 DNA to insect cells in order to prepare a transgenic virus HcNPV carrier to deliver the foreign gene lacZ gene to normal virus -Schematic diagram showing the manufacturing process of HcNPV.

Figure 6 shows the result of confirming that the lacZ gene is inserted into the recombinant virus lacZ-HcNPV genomic DNA by Southern blotting.

7 is a graph showing the production of beta-galactosidase produced by the recombinant virus lacZ-HcNPV over time.

The present invention comprises culturing a Sptopera prujiferda cell line, infecting the cells with white bull moth polyhedron virus (HcNPN DNA), and then multiplying and collecting only the proliferated viral DNA; Cloning the purified HcNPN DNA with an EcoRI restriction enzyme to clone the EcoRI fragment into an EcoRI site of the carrier pUC8 to produce a recombinant carrier pHE-H; The fragment obtained by restriction enzyme treatment of the pHE-H recombinant carrier was inserted into pBluescript SK (+) plasmid to obtain recombinant plasmid pBH2.3, and then introduced into E. coli, and transformed. The pBH2.3 plasmid DNA obtained at this time was used as a template. PCR was carried out using two primers containing restriction enzyme cleavage sites, and then PCR product DNA was inserted into the PHE-H recombinant plasmid after restriction enzyme treatment to prepare a delivery carrier pHcEV and transformed E. coli into this carrier. Extracting DNA after conversion to identify DNA cloned on agarose gel; PHcEVI was prepared by removing two BamH I cleavage sites of the prepared pHcEV carrier, respectively, by BamH I, followed by removal of DNA fragments between EcoR I- xho I sites of the pHcEVI carrier to prepare pHcEVII, followed by pUC8 at pHcEVII. removal of the polyclonal integration sites (MCS) of origin after producing the pHcEVⅢ manufactured pHcEVIII in HcNPV DNA fragment and pBluescript SK (+) MCS and the T3 primer binding portion of Pvu ⅱ with Kpn I enzyme DNA fragment obtained by treating the carrier And preparing a pHcEV-IV carrier obtained by linking a DNA fragment subjected to restriction enzyme treatment with pHcEVIII to introduce a polyA signal of the polyhedrin gene. treating the pHcEV-IV carrier followed by dideoxy nucleotide chain termination to identify polycloning sites in the pHcEV-IV carrier; insert pCH110 a restriction enzyme Kpn Ⅰ and BamH lacZ gene DNA a Ⅰ restriction enzyme treatment to pHcEV-Ⅳ and the double-cut the carrier pHcEV-Ⅳ with Hind Ⅲ and BamH Ⅰ separate the poly A signal and the T3 primer attachment site fragment and again The pHcEV-IV carrier was digested with Kpn I and Hind III to connect the two DNA fragments to prepare recombinant pHcEV-IV-lacZ, and the recombinant pHcEV-IV-lacZ was transformed into E. coli , followed by restriction of MCS. Screening clones that were not cleaved with Nco I restriction enzymes by enzymatically cleaving Nco I enzymes and filling them with cleno fragments; Preparing a recombinant virus lacZ-HcNPV by replacing the lacZ gene in pHcEV-IV and the polyhedrin gene in normal HcNPV by cotransfection into an insect cell of Sptopera prosiferda cells; Culturing the Spododotera luciferda cells, inoculating the virus solution, inoculating and culturing to isolate the virus into which the lacZ gene is inserted, and confirming the insertion of the lacZ gene by Southern blotting; And measuring beta-galactosidase activity, the lacZ gene product produced by the recombinant virus lacZ-HcNPV.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Gene Cloning Delivery Carrier and Recombinant Expression Virus Carrier for Eukaryotic Cells

Step 1: propagation of virus and purification of DNA

Spodoptera frugiperda cell line (IPLB-SF21) was incubated in TC-100 medium containing 10% fetal bovine serum (FBS), followed by Hyphantria cunea nuclear polyhedrosis virus. HL-2: HcNPV) was proliferated by infecting at 0.1 to 5 plaque forming units (pfu / cell) per cell. After 72 hours, the proliferated cells were collected, sonicated for 1 minute, the pulverized cells were centrifuged at 5000 x g for 30 minutes, and the supernatant virus solution was centrifuged at 100,000 x g to precipitate the virus. The precipitated virus was suspended in 500 µl distilled water in an Eppendorf tube, left for 20 minutes, centrifuged at 4000 xg for 10 minutes, and the supernatant was transferred to a new Eppendorf tube. (SDS) was added and the proteinase K was treated at 500 µg / mL and left at 37 ° C for 1-2 hours. Centrifuge at 6,000 xg for 5 minutes to obtain supernatant, and process phenol: chloroform: isoamylalcohol (25: 24: 1) in this order to remove protein, add 95% ethanol twice, mix well, and then at -20 ℃ for 24 hours. Let it stand. The solution was centrifuged at 12,000 × g for 15 minutes at 4 ° C. to collect viral DNA, and the precipitated DNA was dried in vacuo and then dissolved in 100 μl Tris (TE) buffer and stored at 4 ° C. for use.

Second process: EcoR I Cloning of DNA Fragments and Identification of Polyhedrin Genes

The HcNPV DNA purified in the first step was digested with EcoR I restriction enzyme, followed by electrophoresis, and HcNPV fragments were Southern blot (Southern, J. Molecular Biology 98: 503-517, using a polyhedrin gene of AcMNPV as a probe). 1978) showed that the fragment with the polyhedrin gene was an EcoRI-H fragment having 7300 base pairs, and as shown in FIG. 1, the DNA fragment was cloned into the EcoRI region of the carrier pUC8 and named as pHE-H. Carriers were made (Lee et al., J. Kor. Soc. Virology 21: 35-40, 1991).

Third Step: Preparation of pHcEV Delivery Carrier

Cleavage of pHE-H recombinant vehicle (Lee et al., J. Kor. Soc. Virology 21: 35-40, 1991), in which HcNPV EcoRI-H fragment (7.3 kb) was cloned into pUC8 carrier, was digested with Xho I and BamH I restriction enzymes As shown in FIG. 1, a 2.3 kb fragment including a polyhedrin gene promoter and a polyhedrin gene portion was obtained and inserted into the Xho I and BamH I restriction sites of the pBluescript SK (+) plasmid. The total ligation amount was 50 μl T4 DNA ligase and ATP addition ratio was 0.5 mM per unit. Into the Eppendorf tube, 5 μl (1 μg / μl) of the restriction DNA digested vehicle and 20 μl (0.7 μg / μl) of 2.3 kb DNA were added, 5 μl of 5 mM ATP and ligation buffer (66 mM Tri-HCl, pH7.6). , 5 μl of 6.6mMMgCl 2, 10 mM dithiothreitol) and 5 μl of T4 DNA ligase (1.8 unit / μl) were added and reacted for 18 hours at 18 ° C., followed by electrophoresis with 1% agarose gel. The recombinant plasmid was named pBH2.3 and transformed into Escherichia coli E. coli XL-1 blue by the method of Mandel & Higa (J. Mol. Biol. 53: 154-162, 1970), and PCR. It was used as a template DNA for the polymerase chain reaction (a technique for amplifying and synthesizing DNA).

Two initiators (primer-1, 5'-GACGGTACCAATAATCCATGGTATTTATAGGT-3 ') and T3 primers were used to replicate the polyhedrin gene in the pBH2.3 plasmid. Using the PCR mutagenesis of the translation initiation ATG of the polyhedrin gene to a password and ACC, the Nco I restriction enzyme site sequence (CCATGG) and the Kpn I site sequence (GGTACC) by inserting the two bodies -1 was prepared. For PCR amplification, 200ng of pBH2.3 plasmid DNA, 100pmol of T3 primer and primer-1, and Taq polymerase 2unit were added as template DNA. PCR performed 30 cycles of denaturation, association and extension. The first cycle was performed at 94 ° C for 7 minutes, at 55 ° C for 5 minutes, and at 72 ° C for 2 minutes. The remaining cycles were performed at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 3 minutes. After 10 minutes at ℃ was treated with phenol 2 times, treated with chloroform: isoamyl alcohol (24: 1) once, and then added 95% ethanol 2.5 times, and the DNA was precipitated at -70 ℃ 20 minutes. Washed with 75% ethanol, dried, dissolved in 20 μl TE buffer, developed for 30 minutes at 100 volts using 1.0% agarose gel, and then dyed for 15 minutes in 0.5 μg / mL ethidium bromide aqueous solution. It observed and confirmed on the back. Cutting the mutated PCR product DNA with restriction enzymes Xho I and Kpn I enzymes and then, as shown in Fig. 1 were also cloned into the Xho I and Kpn I enzyme site of the recombinant plasmid and pHE-H la pHcEV this naming, E.coli XL1-blue was transformed. DNA was extracted, double cleaved by Xho I and Kpn I enzymes, and digested with Nco I restriction enzymes, and cloned on 0.7% agarose gel.

Fourth Step: Modification of pHcEV Carrier and Preparation of pHcEV-IV Carrier

In this process, a process of making various carriers by modifying the pHcEV carrier is shown in FIG. 2. There are two BamH I sites in the HcNPV DNA fragment downstream of the promoter of the pHcEV carrier. This site is rarely needed and is cleaved with BamH I and removed as shown in FIG. 2, and the remaining large fragments are treated with Klenow fragments to form blunt ends and self-linked to prepare pHcEVI. Next, in order to remove DNA fragments between HcNPV EcoRI-XhoI sites, which are not required upstream of the promoter of pHcEVI carriers, treatment with EcoR I and Xho I restriction enzymes was carried out to treat flat fragments to make flat ends, thereby preparing pHcEVII. In order to remove the polyclonal insertion site (MCS) of the pUC8 source present in pHcEVII, after treatment with BamH I and Nde I, the Klenow fragment was treated to make a flat terminal and self-linked to prepare a 6.1 kb pHcEVIII. In order to insert a multi-cloning site in pHcEVⅢ recombinant carrier pBluescript SK (+) by MCS and the T3 primer binding portion of Pvu Ⅱ and electricity in after processing the Kpn I enzymes and 1.2% agarose gel electrophoresis of the carrier 380bp (bp) Size of The fragments were separated, put into a dialysis tube containing 0.5X TBE buffer, and electrophoresed at 100 V 60 mA to separate DNA. the pHcEVⅢ in order to put a poly A signal (poly A signal) of the polyhedrin gene to the Hind Ⅲ pHcEVⅢ carrier to the enzyme Kpn I cloning a 500bp (bp) separated by a double-cut pUC18 to pUC18-500 made recombinants. This was again cut into EcoR I and treated with Clenow fragment to make a flat end, and again with Hind III to extract electrically. The three prepared DNA fragments were mixed together and reacted with T4 DNA ligase at 18 ° C. for 12 hours to prepare a pHcEV-IV recombinant carrier.

Step 5: Identification of Multiple Cloning Sites in the pHcEV-IV Carrier

As a result of treating each of the restriction sites with the restriction enzymes to confirm whether the multicloning sites inserted into the pHcEV-IV carrier completed in the fourth step were correct, the restriction sites of 13 restriction enzymes were shown in FIG. It was confirmed that they have one by one. In order to reconfirm this, the nucleotide sequence determination of the MCS region was carried out using a Tide primer by a dedeoxynucleotide chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977). The determined sequence is shown in FIG. 3.

Step 6: Preparation of Recombinant Expression Virus Carrier

(1) Preparation of Recombinant Carrier pHcEV-IV-lacZ

Polyhedrin genes were removed by allelic recombination in order to express the normal white bull moth nuclear polyhedron virus HL-2 (HcNPV) genome. It is also necessary to introduce a system for searching for clones of foreign genes. Therefore, in the present invention, the beta-galactosidase gene (lacZ gene) was selected to utilize the above two functions. As shown in Fig. 4 pCH110 (Possee & Howard, Nucleic acid Research 15: 10233-10249, 1987) to Kpn I and Kpn I and BamH I restriction of the lacZ gene DNA was separated by treatment with a restriction enzyme BamH I-IV pHcEV It was transferred to the enzyme site. Carrier pHcEV-IV the Hind Ⅲ and the double-cut with BamH I poly A signal and T3 separating the fragments of the primer attachment site, and again the pHcEV-IV carrier was treated with Kpn I and Hind Ⅲ enzyme the two DNA fragment T4 DNA A recombinant prepared by linking with a ligase was prepared and named pHcEV-IV-lacZ, and the recombinant was transformed into E. coli XL1-blue. In order to match the translational initiation code of the downstream promoter of pHcEV-IV-lacZ with the lacZ gene cloned with the initiation code of the lacZ gene, NcoI enzyme, a restriction enzyme cleavage site of the MCS, was treated and filled with a Klenow fragment. Clones that were not cleaved with Nco I restriction enzyme were selected.

(2) Preparation of Expression Recombinant Virus

In order to recombinantly replace the lacZ gene in the recombinant pHcEV-IV-lacZ and the polyhedrin gene in the normal HcNPV, cotransfection was carried out to the insect cells, Spododoter luciferda cells, as shown in FIG. 5. Inoculate 1 x 10 ⁶ insect cells in two 35 mm Petri dishes and incubate for 12 hours at 28 ° C. Remove the medium, wash the cells with 2mL TC-100 base medium, and discard 2mL TC-100 base medium. It was incubated for 30 minutes at room temperature. Meanwhile, 10 μl of pHcEV-IV-lacZ DNA solution (100 ng / μl), 20 μl of HcNPV DNA solution (100 ng / μl) and 20 μl of distilled water were added to a sterile polystyrene tube, and 11 μl lipofectin ( lipofectin, 1.0 mg / mL, Bethesda Research Laboratory, USA) and 50 µl of 99 µl sterilized tertiary distilled water were mixed and left at room temperature for 15 minutes to form liposomes. Remove the medium of Petri dish and put 1.5mL TC-100 medium again, add the DNA-lipopeptin mixture and shake the Petri dish and incubated for 5 hours at 28 ℃. In addition, 1.5mL TC-100 (containing 10% fetal bovine serum and antibiotics) was added and incubated at 28 ° C. for 72 hours. After incubation, the virus-containing medium was plaque analyzed to select recombinant virus lacZ-HcNPV. The process is shown in FIG.

Step 7: Selection and Identification of Recombinant Virus

Dispense 2 x 10 ⁶ cells / mL of Spodoptera frugiperda cells into a cell culture petri dish (60 x 15 mm), incubate about 5 mL of TC-100 medium, and incubate at 28 ° C. Incubate for about an hour. After 24 hours, the medium was drained, 0.1 mL of the virus solution was inoculated in a petri dish, and infected with shaking at 28 ° C. for 60 minutes at 15 minute intervals. After 60 minutes, the supernatant on the cells was washed with phosphate buffer, X-gal mixed with 0.8% low melting temperature agarose to a final concentration of 200 μl / mL, and then covered with infected cells in a Petri dish. Blue plaques were selected at 12 hour intervals after 24 hours by incubation at 28 ° C. The blue plaque means that the lacZ gene is inserted into the virus and the gene is expressed. The white plaque means normal virus without the gene. The recombinant virus that formed the blue plaque was isolated and confirmed by the Southern blot method (Southern, J. Mol. Biol. 98: 503-517, 1975) using a probe to determine whether the lacZ gene was inserted into the virus. . This is shown in FIG. Recombinant virus was isolated from the proliferation of insect cells, the DNA was purified by the method described in the first step, then digested with restriction enzymes and developed by electrophoresis, followed by Southern blotting using a probe. Plate A of Figure 6 is a recombinant virus DNA digested with restriction enzyme, plate B means a DNA fragment inserted lacZ gene using a probe. Insertion of the lacZ gene into the recombinant virus was observed in lanes 3 ', 4' and 5 'of plate B. The results of FIG. 6 clearly demonstrate that the recombinant virus was produced and the recombinant virus identified in the present invention was named lacZ-HcNPV and was deposited with the KFCC 11109 on October 21, 1999 at the Korean spawn association association microbial deposit center.

Example 2: Recombinant Protein Production in Insect Cells

In this embodiment, the beta-galactosidase activity, a lacZ gene product produced by the recombinant virus lacZ-HcNPV prepared in Example 1, was used in Miller method (Miller, Experiments in molecular genetics, pp352-356, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972). The activity of an enzyme capable of degrading 1.0 nmol of ONPG (o-nitrophenyl-β-D-galactopyranoside, Sigma) for 1 minute was defined as 1.0 unit. 2 × 10 ⁶ cells were lysed in 100 μl of 0.25 M Tris-HCl (pH8) and sonicated three times for 15 seconds at 100 μA intensity using an ultrasonic grinder (US600, Nissei). Cell lysates were centrifuged at 10,000 × g for 5 minutes and 100 μl of supernatant was used to measure beta-galactosidase activity. 1.0 mL of Z-buffer (0.1 M sodium phosphate buffer; pH7.5, 1 mM MgCl2, 45 mM β-mercaptoethanol) was added to 30 µl of the supernatant, and pre-heated at 28 ° C, followed by ONPG (4.0 mg / ml). , Sigma) was added and incubated at 28 ° C. for 20 minutes, and the absorbance at 420 nm was measured using a 420 nm spectrophotometer (UV-240, Shimadzu). Cell samples were collected at 24, 48, 72, 96 and 120 post-infection and examined for activity and maximum increase. The enzyme activity was calculated by the following equation.

As a result, Figure 7 shows the amount of beta-galactosidase produced by the recombinant virus lacZ-HcNPV and production continues to increase from 24 hours after infection, 50 UI / min / mL at 48 hours, 170 UI at 120 hours / min / mL is produced, indicating that the recombinant virus expresses the lacZ gene. Recombinant virus prepared in the present invention was found that the foreign gene is inserted into the lacZ gene region, and the currently inserted lacZ gene is expressed as a foreign gene, it was found that the practicality is very high.

As described in the above embodiment, the polyhedrin gene is expressed by a delivery carrier-expressing virus carrier-host system that expresses a foreign gene using an insect cell that is propagated by the phantom tria cunea nuclear polyhedrosis virus. It is a very useful invention in the basic biotechnology industry related to genetic recombination and protein production because it has an excellent effect of enabling the mass production of proteins using a promoter.

Claims (5)

Recombinant eukaryotic gene transfer carrier pHcEV-IV containing at least 13 restriction enzyme cleavage sites by cloning a DNA fragment containing a polyhedrin gene promoter of white moth polyhedron virus (HcNPV) into a cloning carrier of Escherichia coli. Recombinant expression virus carrier prepared by replacing the polyhedrin gene in the virus with a foreign gene carried by the recombinant eukaryotic cell gene delivery vehicle of claim 1 foreign gene-HcNPV. The recombinant expression virus carrier lacZ-HcNPV (Accession No. KFCC 11109) according to claim 2, wherein the foreign gene substituted with the polyhedrin gene is a beta-galactosidase gene (lacZ). Recombinant protein production method characterized by introducing the foreign gene into the recombinant expression virus carrier foreign gene-HcNPV described in claim 2 using the recombinant eukaryotic cell gene delivery carrier pHcEV-IV described in paragraph 1 and then cultured in insect cells . The method of claim 4, wherein the insect cells are Spodoptera frugiperda cell line (IPLB-SF21) cells.