KR20080059702A - Hr3 enhancer from bombyx mori nucleopolyhedrovirus k1 and construction of method for mass production of a recombinant protein using hr3 - Google Patents

Abstract

A hr3(homologous region 3) enhancer isolated from Bombyx mori nucleopolyhedrovirus K1 is provided to maximize the activity of promoter and produce a large quantity of a recombinant protein by optimizing the location and direction of the enhancer in the expression vector. A hr3 enhancer isolated from Bombyx mori nucleopolyhedrovirus K1 has the nucleotide sequence of SEQ ID NO:1. The mass-production of a recombinant protein is accomplished by locating the hr3 enhancer of SEQ ID NO:1 in a site downstream of the SV40 poly A site in 3' to 5' direction in a protein expression vector.

Description

Hr3 enhancer from Bombyx mori nucleopolyhedrovirus K1 and construction of method for mass production of a recombinant protein using hr3}

FIG. 1 shows the results of nucleotide sequence analysis of the hr3 region separated from BmNPV K1, followed by homology analysis of the previously known hr3 region (FIG. 1A shows the nucleotide sequence of the hr3 region separated from BmNPV K1). 1B and 1C show the homology between the hr3 nucleotide sequence of BmNPV K1 isolated in this study and the three previously identified BmNPV hr3.BMU77353, BMU51238 and L24902 are the three hr3s registered in GenBank. BmNPV K1 represents the hr3 of BmNPV K1 isolated in the present invention).

2A is given to the Kpn Ⅰ, Nhe Ⅰ, BamH Ⅰ and Sal Ⅰ recognition sequence in 5 '→ 3' direction and a 3 '→ 5' direction of the separation hr3, Kpn Ⅰ- 5 '→ 3'- Nhe Ⅰ ( U-53), Kpn I-3 '→ 5'- Nhe I (U-35), BamH I-5' → 3'- Sal I (D-53) and BamH I-3 '→ 5'- Sal I (D-35) Hp32 of the pGL3-Hp32 transporter containing four fire hr3 insert genes containing a firefly luciferase gene downstream of the silkworm hypothetical protein 32 (Hp32) promoter as a marker gene. Carriers produced upstream of the promoter and downstream of the SV40 Poly A were constructed with pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53 and pGL3-Hp32-D35 carriers. FIG. 2B shows a standardized carrier pRL-A3-R luc for normalization of four pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53, and pGL3-Hp32-D35 carriers. It is.

Figure 3 shows the results of analysis of luciferase activity against the control carrier pGL3-Hp32 and four types of pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53 and pGL3-Hp32-D35 carrier.

The present invention relates to the optimal introduction position and the introduction direction of the expression carrier of the hr3 enhancer isolated from the silkworm polyhedral virus

Homologous regions (hrs) of baculovirus refer to regions having repeated sequences in the genome of baculovirus. To date, homologous regions of baculovirus are known as Bombyx Autographa including mori Nucleopolyhedrovirus (BmNPV) californica NPV (AcNPV), Lymantria dispar NPV (LdNPV), Orgyoa psudotsugagta NPV ((OpNPV), etc. (Lu et al., 1997) .There are seven hr1, hr2L, hr2R, hr3, hr4L, hr4R and hr5 in the BmNPV T3 genome (Gomi et al., 1999). Most of these homologous regions have been shown to play a very important role in viral DNA replication, as well as to function as enhancers for transcription of viral and nonviral genes (Lo et al., 2002, Viswanathan et al., 2003) Therefore, much research has recently been conducted to use this hr3 as an enhancer for expression of recombinant proteins, and it has been substantially improved compared to a vehicle which does not introduce hr3 by introducing hr3 in a protein expression carrier. Excess marker genes were identified (Gray et al., 2004, Tang et al., 2005, Nagamine et al., 2005).

However, there are no studies on how the hr3 enhancer can maximize the activity of the promoter when introduced into which position in the expression carrier for expression of useful recombinant protein.

The present inventors have completed the present invention by determining the optimal introduction position and introduction direction in the protein expression carrier for the hr3 region isolated from BmNPV K1, thereby improving the expression of recombinant protein about three times as compared to the carrier without introducing the hr3 region. It became.

Accordingly, it is an object of the present invention to provide an optimal introduction position and introduction direction in a protein expression carrier for the hr3 region of BmNPV K1.

In order to determine the optimal introduction position and introduction direction in the expression carrier of the hr3 enhancer isolated from silkworm polyhedron virus (BmNPV K1),

1) synthesizing primers for the hr3 region of the previously isolated BmNPV T3 strain, amplifying the hr3 region of BmNPV K1 by performing PCR using the genome of the BmNPV K1 strain as a template;

2) preparing a pGEMT-hr3 carrier by cloning the hr3 region of BmNPV K1 amplified in step 1) into a pGEM-T easy carrier (Promega, USA),

3) analyzing the entire base sequence for hr3 in the pGEMT-hr3 carrier prepared in step 2),

4) The pGL3-Hp32 carrier (Lucifer Bioinformatics Lab.) Containing the luciferase gene as a marker gene downstream of the hypothetical protein 32 (Hp32) promoter of the silkworm in the hr3 region of BmNPV K1 isolated from step 3). Silkworm Hp32 promoter upstream and downstream of SV40 poly A in the 5 '→ 3' and 3 '→ 5' directions, respectively, for pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53 and pGL3- Fabricating the Hp32-D35 carrier, and

5) Luciferase activity against each of the four pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53 and pGL3-Hp32-D35 carriers prepared in step 4) and the control carrier pGL3-Hp32 carrier By the method comprising the, characterized in that for determining the optimal introduction position and introduction direction in the expression carrier of the hr3 enhancer isolated from silkworm polyhedron virus (BmNPV K1).

In order to determine the optimal introduction position and the direction of introduction in the expression carrier of the hr3 region developed in the present invention, the silkworm Hp32 of the pGL3-Hp32 carrier containing a luciferase gene downstream of the hypothetical protein 32 (Hp32) promoter of silkworms. Hr3 is introduced in the 5 '→ 3' and 3 '→ 5' directions upstream of the promoter and downstream of the SV40 poly A, respectively, for pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53 and pGL3-Hp32-D35 The carrier was produced. Four different carriers, including the control vehicle, pGL3-Hp32, were compared for luciferase activity using the Dual Luciferase Assay System (Promega, USA).

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the protection scope of the present invention is not limited to the following examples.

Example  One. BmNPV K1 of hr3  Isolating Regions and Decoding Sequences

To isolate the hr3 region of the baculovirus BmNPV K1 strain, a forward primer (5'-ACATTTTATGTCGAAAACAAATGAC-3 ') and a reverse primer (5'-CATATGAGCAGACCCGTCTTGGCGA-3) for the hr3 region of the previously identified BmNPV T3 strain genome ') Was synthesized respectively. The synthesized forward and reverse primers were used for PCR reactions based on the genome of the BmNPV K1 strain. The PCR product amplified by the PCR reaction was cloned into a pGEM-T easy carrier to prepare a pGEMT-hr3 carrier, followed by full sequencing of the hr3 region (see FIG. 1). As shown in FIG. 1B, the hr3 nucleotide sequence of the BmNPV KI strain (Korean species) shows a sequence difference from the previously reported Japanese and Chinese species.

Example  2. Separated BmNPV K1 hr3 Four Carriers for the Determination of Optimal Positions and Directions for Protein Expression Carriers

The terminal sequence of the embodiment 15 of the BmNPV K1 hr3 isolated from '→ 3' direction and a 3 '→ 5' direction Kpn Ⅰ (GGTACC), Nhe Ⅰ (GCTAGC), Bam HⅠ (GGATCC) and Sal Ⅰ (GTCGAC ) Flanking was used to synthesize forward and reverse primers. Each of the forward and reverse primers synthesized was used for PCR reaction with pGEMT-hr3 as a template. Amplified by the PCR reaction Kpn Ⅰ- 5 '→ 3'- Nhe Ⅰ (U-53), Kpn Ⅰ- 3' → 5'- Nhe Ⅰ (U-35), Bam HⅠ- 5 '→ 3'- Sal PCR products of I (D-53) and Bam HI-3 '→ 5'- Sal I (D-35) were cloned into pGEM-T easy carriers, respectively, for pGEMT-U53, pGEMT-U35, pGEMT-D53 and pGEMT- D35 was produced. PGEMT-U53 and pGEMT-U35 were treated with restriction enzymes Kpn I and Nhe I, respectively, and then pGL3 containing a firefly luciferase gene downstream of the silkworm hypothetical protein 32 (Hp32) promoter. respectively introduced between the carrier -Hp32 Hp32 within the Kpn ⅰ and Nhe ⅰ restriction position upstream of the promoter, to prepare a pGL3-U53-U35-Hp32 Hp32 and pGL3-carriers of two or more. The remaining two of pGEMT-D53 and pGEMT-D35 are respectively introduced between the respective Bam HⅠ and Sal Ⅰ in one process at the same time and then, pGL3-Hp32 carriers within the SV40 poly (A) downstream of the restriction endonuclease site is Bam HⅠ and Sal Ⅰ, pGL3 -Hp32-D53 and pGL3-Hp32-D35 were constructed (see Figure 2A).

On the other hand, the production of standardized carriers for normalization of the four types of carriers (pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53, and pGL3-Hp32-D35) were used as marker genes. In the pRL-SV40 carrier (Promega, USA) containing the Renilla luciferase gene, the SV40 promoter was removed by restriction enzymes Kpn I and Hind III, and then the silkworm actin 3 (A3 cytoplasmic actin gene) promoter was introduced at that position. Thus, pRL-A3-R luc , a standardized carrier capable of standardizing firefly luciferase activity, was prepared (FIG. 2B).

Example  3. pGL3 - Hp32  Carrier By contrast  4 kinds pGL3 - U53 - Hp32 , pGL3 - U35 - Hp32 , pGL3 - Hp32 - D53  For each of the and pGL3-Hp32-D35 carriers Luciferase  Activity analysis

Lucifer for four carriers, pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53, pGL3-Hp32-D35, and the control carrier pGL3-Hp32, each having a different introduction position and introduction direction. Raise activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega, USA). In other words, 400 μl of pGL3-hp32, pGL3-U53-Hp32, pGL3-U35-Hp32, pGL3-Hp32-D53, and pGL3-Hp32-D35 carriers and 40 ng of standardized carrier, pRL-A3-R luc 40 ng, were added to 25 μl of sterile distilled water. 2 μl of FuGENE HD Transfection Reagent (Roche, USA), an intracellular nucleic acid introduction reagent, was mixed again and treated with Bm5 silkworm cultured cell lines grown in 24 well plates. After 48 hours of treatment, the culture medium was removed, washed with 1X PBS solution, and 100 µl of 1X Passive Lysis Buffer was added thereto, followed by rotary stirring for 15 minutes to decompose the cultured cells.

Dispense 100 μl of 1 × Firefly Luciferase Assay Reagent II into 5 wells on a 96 well white plate (Greiner bio-one, Germany), mix 20 μl of each cell lysate prepared above in each well, and Firefly luciferase activity was measured for four hr3 carriers, including the control vehicle, using a luminometer (TECAN, USA).

To normalize Firefly luciferase activity, immediately after firefly luciferase activity measurement, 100 μl of 1X Stop & Glo Subtrate, a firefly luciferase activity inhibitor and Renilla luciferase activity assay solution, was added, followed by a luminometer (TECAN, USA ), Renilla luciferase activity was measured for four hr3 carriers, including each control vehicle. Luciferase activity measurements were performed in three replicates.

Firefly luciferase measurement for each of the carrier measured by a Luminometer is a Renilla measurement for each carrier The normalization was divided by the luciferase measurement.

At this time, the normalized value of the control carrier pGL3-Hp32 was set to 100, and then calculated as relative values for the four hr3 carriers. As shown in FIG. 3, the relative value of the pGL3-Hp32-D35 carrier was 287.1%, and the relative values of pGL3-U53-Hp32, pGL3-U35-Hp32, and pGL3-Hp32-D53 were 150.9% and 145, respectively. % And 171.8% were relatively low.

Therefore, the optimal introduction position and direction in the expression carrier of hr3 enhancer isolated from silkworm polyhedron virus will be able to produce the most excess recombinant protein when hr3 region is introduced downstream of SV40 poly A in 3 '→ 5' direction. Judging.

The hr3 isolated from the silkworm polyhedron virus (BmNPV) has been confirmed to function as an enhancer and has been used as an enhancer for promoters for the expression of useful recombinant proteins. However, no studies have been conducted on how the hr3 enhancer can maximize the activity of the promoter when introduced into which position in the protein expression carrier for expression of useful recombinant protein. Therefore, the determination of the optimal introduction position and introduction direction in the protein expression carrier of the hr3 region developed in the present invention will be usefully used to produce more useful recombinant protein in the same effort.

<110> Rural Development Administration <120> Optimal location and direction of hr3 enhancer from Bombyx mori          nucleopolyhedrovirus in expression vector <130> 1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 461 <212> DNA 213 hr3 from Bombys mori nucleopolyhedrovirus <400> 1 acattttatg tcgaaaacaa atgacatcag cttatgattc atacttaatc gtgcgttaca 60 agtagaattc tactcgtaaa gcgagtttaa tttgaaaaac aaattagtca ttattaaaca 120 tgttaacaat cgtgtataaa aatgacatca gtttaatgat gacatcatct cttgattatg 180 ttttacacgt agaattctac tcgtaaagcc agttcagttt tgaaaaacaa atgacatcat 240 ctttcgatta tgttttacac gtagaattct actcgtaaag ccagttcagt tttgaaaaac 300 aaatgacatc atttcttaaa ttcggttttg aaaaacaaat gacatcatct cttgattgtg 360 ttttacacgt agaattctac tcgtaaagcc agttcaattt tgaaaaacaa atgacatcat 420 cttagaggta gactcgtcgc caagacgggt ctgctcatat g 461  

Claims (3)

Nucleotide sequence of the hr3 enhancer isolated from the silkworm polyhedral virus (BmNPV K1 strain) described in SEQ ID NO: 1. A method for producing a recombinant protein by using the hr3 enzyme of claim 1 as an appropriate introduction position and introduction direction in a protein expression carrier. The method of claim 2, wherein the introduction direction and introduction position of the hr3 enhancer is introduced downstream of the SV40 poly A in a 3 ′ → 5 ′ direction.

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