KR101460861B1 - Expression of bovine lactoferrin using transformed Chlorella - Google Patents

Abstract

The present invention relates to a method for expressing a lactoferrin protein from a dairy cow using microalgae chlorella, more precisely by introducing a lactoferrin gene optimized with a codon suitable for chlorella into an expression cassette containing a dual CaMV35S promoter and expressing the cassette in an expression vector To construct a recombinant lactoferrin expression vector. And a method for expressing a recombinant lactoferrin protein by transforming the thus-constructed expression vector into chlorella.
If a large amount of chlorella containing the recombinant lactoferrin expression system is cultured according to the present invention, the production of recombinant lactoferrin is increased, so that it can be easily used as an antibiotic substitute for animals.

Description

Expression of bovine lactoferrin using transformed Chlorella using the transformed chlorella.

The present invention relates to the expression of recombinant lactoferrin from cow's milk in microalgae. More specifically, the present invention is characterized by expressing recombinant lactoferrin by transforming a cow-derived lactoferrin gene into chlorella with a codon-optimized lactoferrin gene suitable for chlorella nucleotides Lt; / RTI >

Lactoferrin is a type of transferrin, a protein that binds iron, and is mainly found in milk, milk, blood, saliva, tears, mucus secretions, and the like. The first lactoferrin was purified from bovine milk, and soon it was separated and purified in the milk of several mammals. When lactoferrin was first discovered, it was called 'red protein' in the sense that it was a reddish protein. This red color appears as a result of binding of lactoferrin to iron. The lactoferrin that is not bound to iron ion is colorless, and the property of lactoferrin bound to iron is a very important characteristic in the function of this protein in vivo. Lactoferrin derived from a cow is a glycoprotein with a molecular weight of about 70,000. It has two leaf-like lobes like ginkgo bilob, slightly overlapping each other, and each lobe has one binding site that binds with iron. Therefore, one molecule of lactoferrin is bound to the iron ion. The binding of lactoferrin to iron ion is about 260 times stronger than that of transferrin, and this property of lactoferrin has an essential function in capturing iron rather than the transport of iron.

 It has been reported that lactoferrin is an antimicrobial activity that is representative of the biological activity of lactoferrin, and it is reported to be effective in various aspects such as antiviral action, anti-cancer activity, anti-inflammatory action (YK Rye and WS Kim. Dairy Sci. Technol., Vol. 27 (1), pp. 19-28). Lactoferrin, which has such a good function, has a high content in lactose, and is most abundant in colostrum, which is a yellowish and diluted milk which is secreted for a few days after labor. It is contained in human colostrum 6 to 8 mg / The breast milk contains about 2mg / l. Colostrum of milk contains 1.2mg / ℓ and 0.1 ~ 0.2mg / ℓ in general milk. Currently, lactoferrin is purified from milk and used.

 Because of the very low production of this multifunctional lactoferrin, research and development has been conducted to produce recombinant proteins of this protein. There is a method of expressing human lactoferrin by recombinant lactoferrin from a mammal (Korean Patent Publication No. 0192718) and a method of expressing human lactoferrin using insect cells (Korean Patent Publication No. 0331981), but the cost for producing recombinant lactoferrin It is not economical because it is high. Microorganisms have also been used to express recombinant lactoferrin. (Korean Patent Publication No. 0543462) using a prokaryotic Bacillus strain (Korean Patent Publication No. 0543462), a method for producing a recombinant human lactoferrin using a fungus aspergillus as a eukaryotic organism (Korean Patent Publication No. 0383436) and a human lactoferrin production using a yeast strain (Korean Patent Publication No. 0468593). Plants were also utilized to express recombinant lactoferrin. A method for mass production of human lactoferrin in plant cell culture (Korean Patent Publication No. 0551713), and a method for producing human lactoferrin using rice (U.S. Patent No. 7,276,646).

Currently, lactoferrin is lactoferrin purified from milk rather than recombinant lactoferrin. First, lactoferrin is used for a variety of applications, but because the production is too small, various methods have been tried to increase the production of lactoferrin.

US 0014797 A1

 Si-Shen Li, Huai-Jen Tsai. Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract. Fish & Shellfish Immunology 2009; 26; 316-325

Most of the commercial commercially available recombinant proteins are produced using bioreactors designed for the cultivation of bacteria, yeast or animal cells. Protein expression systems by bacteria are lacking in transcriptional or post-translational coordination processes such as splicing, glycation, and protein assembly. In addition, there is a high possibility that bacterial endotoxin and protease and other impurities are mixed. Eukaryotic expression system by yeast does not faithfully realize glycosylation process. Currently, most of the medical proteins are produced through animal cell cultivation, but mass production is not easy, the medium is expensive, is easily contaminated, and the expression amount is low and the production cost is high. Transgenic animals cost up to $ 500,000 per production, require labor-intensive management, and require a long time to mass-produce.

      Transgenic plants are an expression system with relatively many advantages for the mass production of proteins with stability and functionality. For commercialization of vaccines, antibodies, enzymes, hormones, and medical proteins, the expression level should be at least 5% of the total amount of water-soluble proteins in the cell, generally 0.00146.1%. In general, the major human pathogens, except for some toxic bacteria and fungi, such as Pseudomonas aeruginosa, do not host the plant. Plant viruses do not infect humans or animals, and animal viruses are known to not work on plants. Environmental pollution caused by genetically modified plants There are also problems such as an enzyme reaction on plant components, contamination of proteins, regulation of permission of pharmaceutical proteins.

      Eukaryotic microalgae, which have been developed and studied for over a decade or more, can be an alternative expression system to solve the problems of other strains. Recently, studies on the production of antibodies, insecticides and vaccines by microalgae have been conducted. Microalgae are subject to genetic manipulation for increased productivity of natural ingredients and synthesis of new substances because of the potential for large-scale cultivation under conditions of growth that are not suitable for general crops. Genetic engineering techniques for microalgae are still at a novice level and none of the recombinant products produced from microalgae have been commercialized yet.

In order to accomplish the above object, the present invention provides codon optimization suitable for the chlorella nuclear gene codon in order to express the cow lactoferrin gene in microalgae chlorella. In the codon optimization, the N loop part of the antimicrobial core of lactoferrin optimized 990 bp (LfB-N). The optimized lactoferrin gene was inserted into an expression cassette containing a dual CaMV35S promoter to construct a lactoferrin expression cassette, which was then inserted into the pCAMBIA1304 vector to construct an expression vector. And the expression vector is transformed into chlorella to express the recombinant lactoferrin protein.

The present invention is effective in providing a method for expressing lactoferrin derived from cow in microalgae, Chlorella vulgaris. The present invention constructs an expression vector by inserting a chlorella codon-optimized LfB-N gene into an expression cassette containing a dual CaMV35S promoter in a microalga, chlorella, and inserting it into pCAMBIA1304. This expression vector was transformed into chlorella to express recombinant lactoferrin, and the antibacterial effect was also confirmed. Therefore, it is expected that a large amount of chlorella producing recombinant lactoferrin can be cultured by the method of the present invention, and a new antibiotic substitute can be obtained if an extract is obtained.

Figure 1 shows the expression casstte construction by inserting LfB-N into the pRTL2 vector.
Figure 2 shows the pRTL2-LfB-N library identification by PCR analysis
lane 1: pRTL2, lane 2 to 10: Transformant (pRTL2-LfB-N)
FIG. 3 shows the expression vector (pGG12) construction diagram by inserting an expression cassette into pCAMBIA1304 vector
Figure 4 shows the pGG12 library confirmation by PCR analysis
lane 1: T1, lane 2: T3, lane 3: T4, lane 4: T5, lane 5: T6, lane 6: T7, lane 7: T8, lane8: T9, lane 9: T10, lane 10: 11: T17, lane 12: T20
FIG. 5 is a graph showing the results of screening of chlorella transformants by PCR analysis
lane 1: CT1, lane 2: CT2, lane 3: CT4, lane 4: CT5, lane 5: T6, lane 6: CT7, lane 7: CT9, lane8: CT13
Figure 6 shows SDS-PAGE (A) and immunodetection (B) of recombinant lactoferrin
lane 1: Non-transformant, lane 2: Transformant (pCAMBIA1304)
lane 3: Transformant (pGG12-CN7), lane 4: Transformant (pGG12-CN9)
Figure 7 shows the antimicrobial activity of recombinant lactoferrin

(1) Lactoferrin gene codon optimization

In order to express a recombinant protein by introducing a foreign gene in microalgae, optimizing the codon for the microalgae nucleus gene to be expressed is one of methods for increasing the expression rate of the recombinant protein. For this reason, the cow lactoferrin gene was subjected to codon optimization to suit the chlorella gene. This optimization was commissioned by Biona 's gene synthesis service to optimize the lactoferrin N loop part (990 bp) containing the antimicrobial active core, lactoferricin. Nco I site and start codon (ATG) were inserted at the 5 'end of the gene synthesis, and the gene was synthesized to insert the end codon (TGA) and the Kpn I site at the 3' end. The codon-optimized lactoferrin gene (LfB-N) was cloned into the pGEM T easy vector.

(2) Construction of Expression cassette

LFb-N was isolated from pGEM T easy LfB-N by digestion with Nco I / Kpn I restriction enzyme and purified with gel purification kit. The pRTL2 vector containing the dual CaMV35S promoter was digested with Nco I / Kpn I restriction enzyme to prepare a backbone. LfB-N was inserted into the backbone using T4 DNA ligase to construct pRTL2-LfB-N (FIG. The library was constructed by cloning into E. coli XL1 blue. The constructed library was confirmed by PCR analysis using primer 998-F / 998-R. Table 1 shows the primers used. PCR was carried out at 95 ° C for 2 min, 30 cycles of denaturation at 94 ° C for 1 min, adherence at 64 ° C for 2 min, extension at 72 ° C for 3 min and final expansion at 72 ° C for 7 min. As a result, LfB-N, which is a PCR product of about 1.0 kb, was identified as shown in FIG.

primer The base sequence (5` → 3`) Remarks 998-F ATG GTT CGA TGG TGC ACC ATT TC Start codon 998-R TCA TTC CCT CAG GTT CTT CAG G Stop codon

(1) Construction of expression vector and transformation of Agrobacterium

In order to transform chlorella, the multicloning site of the expression vector pCAMBIA1304 was digested with HindIII / EcoRI restriction enzymes to make a backbone. An expression cassette containing LfB-N as a primer rtl21304-F / rtl21304-R was synthesized by PCR using pRTL2-Lfb-N as a template. Table 2 shows the primers used. PCR was performed at 95 ° C for 2 minutes, 30 cycles of denaturation at 94 ° C for 1 minute, adhesion reaction at 67 ° C for 2 minutes, extension reaction at 72 ° C for 3 minutes, and final expansion reaction at 72 ° C for 7 minutes. The synthesized approximately 2.3 kb expression cassette was purified with a gel purification kit and inserted into the vector via the infusion cloning method in the pCAMBIA1304 backbone (FIG. 3). This expression vector was named pGG12. The constructed vector was cloned into E. coli XL1 blue to construct a library. The confirmation of this library was confirmed by PCR analysis using the primer rtl21304-F / rtl21304-R as a 2.3 kb PCR product. The results are shown in FIG. 4, and T7, 8, 9, 17, and 20 were found to be pGG12. The constructed expression vector pGG12 was transformed into Agrobacterium by electroporation.

primer The base sequence (5 '- > 3') rtl21304-F CCA TGA TTA C GA ATT C GC ATG CCT GCA GGT CAA CAT GG EcoR I rtl21304-R GGC CAG TGC C AA GCT T GG GGA TGT GCT GCA AGG CGA Hind III

(1) Chlorella culture conditions

In the case of chlorella liquid culture, the culture of chlorella seeds was started at a concentration of about 1.0 × 10 ⁵ cfu / ml in the liquid medium of BG11, the light amount was maintained at about 1,000 lux, and the light period of day: night = 16: 8 hours was maintained .

In the case of chlorella solid culture, 0.1 ml of the chlorella culture was dispensed into the BG11 solid medium and plated on the BG11 solid medium. The smoked petri dishes were placed in a clear culture container containing a carbon dioxide gas pack and cultured. The incubation temperature was maintained at 25 ° C, and the light intensity was maintained at about 1000 lux during incubation, and maintained the light period of day: night = 16: 8 hours.

(2) Chlorella transformation

Chlorella transformation is carried out by transforming chlorella using Agrobacterium (Thye San Cha, Willy Yee, Ahmad Aziz (2012). Assessment of factors affecting Agrobacterium-mediated genetic transformation of unicellular green alga, Chlorella vulgarils. World J. Microbiol. Biotchnol. 28: 1771-1779). First, after culturing chlorella 50ml in BG11 liquid medium at a concentration of about OD ₆₀₀ value of 0.7 150 μM acetosyringone (AS) 100 plated on BG11 agar ㎕ chlorella culture medium (pH 5.5) containing the incubation will be for about 3 days. Agrobacterium transformed with pGG12 is cultured simultaneously with chlorella culture. Inoculated with transformed Agrobacterium in 5ml LB liquid medium and cultured OD ₆₀₀ value of about 1.0 at 25 ℃. After completion of the Agrobacterium culture, the cells are recovered by centrifugation, and the recovered cells are suspended in the BG11 liquid medium. About 200 μl of the transformed Agrobacterium suspension is inoculated onto the chlorella solid medium cultured for 3 days and plated. The smoldered solid medium is co-cultivated at 25 DEG C for about 3 days. After co-cultivation, the cells were recovered from the solid medium and recovered by washing with BG11 liquid medium at 12,000 rpm for 10 minutes by centrifugation. Cell washing is carried out about 3 times. The recovered chlorella cells are cultured in a BG11 liquid medium containing cefotaxime (100 μg / ml) at 25 ° C. for 16 hours under dark conditions. After completion of the culture, the cells are inoculated into a BG11 solid medium containing hygromycin (10 μg / ml), a chlorella transformant selection medium, and cultured at 25 ° C. for about 7 to 10 days.

(1) Chlorella transformant Chlorella screening transformed through PCR analysis

The transformed colonies were inoculated into BG11 liquid medium (10 ml) containing hygromycin (10 / / ml) and cultured in the same manner as in Example 3. At a culture concentration of OD ₆₀₀ > 1.0, the culture was centrifuged at 12,000 rpm for 10 minutes to collect the cells. The recovered cells were extracted with DNeasy Plant Mini kit (QIAGEN). The extracted nucleic acid was subjected to PCR reaction with the primer shown in Table 1, and a 1.0 kb band-confirmed colony was selected as a transformant through agarose electrophoresis (FIG. 5). As a result, CT7 and CT9 were identified as transformed chlorella.

(1) Recombinant lactoferrin expression in transformed chlorella

The Hygromycin CT 7 and CT 9 to 50ml BG11 liquid medium supplemented with (10ug / ml) is added 25, light condition is photoperiod 16: cultured at: (night day), the amount of light 100 mol / m ² / s for 7 days 8. The end the culture ^{(1.0 x 10 7 cfu / ml} ) to 12,000 rpm and then the cells were collected by centrifugation for 10 minutes, 2ml total protein extraction buffer (50mM Hepes, 250mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM phenylmethylsulfonyl fluride ). The cell suspension was sonicated (30 W, 10 min) to disrupt the cells and centrifuged at 12,000 rpm for 10 min to recover the supernatant. The recovered supernatant was concentrated using a 10 kDa membrane filter, and 30 ug of total protein was separated by SDS-PAGE (Fig. 6-A). SDS-PAGE revealed a band of approximately 33 kDa on CT7 and CT9.

After western blotting, immunodiplication was performed (Fig. 6-B). Goat anti-bovine Lf (polyclonal) was used as the primary antibody and donkey anti-goat IgG-AP was used as the secondary antibody. As a result, a band of about 33 kDa was confirmed in CT 7 and CT 9.

(1) Biological assay for recombinant lactoferrin

Chlorella transformants CT7 and CT9 containing recombinant lactoferrin were cultured for 7 days in 100 ml of BG11 medium containing hygromycin (10 ug / ml). The cells were recovered by centrifuging the culture (1.0 x 10 ⁷ cfu / ml) at 12000 rpm for 10 minutes. 10 ml of PBS solution was added to the recovered cells, and the cells were washed by centrifugation at 12,000 rpm for 10 minutes. This washing procedure was performed three times. Suspended in 2 ml total protein extraction buffer (50 mM Hepes, 250 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluride). The cell suspension was sonicated (30 W, 10 min) to disrupt the cells and centrifuged at 14,000 rpm for 10 min to recover the supernatant. The recovered supernatant was concentrated with a 10 kDa membrane filter to prepare 500 ug of total protein.

Escherichia coli was selected as a test strain and cultured in 5 ml of LB broth. Chlorella total protein (500 ug) containing recombinant lactoferrin was used and inoculated with 8 mm paper discs. As a positive control, 500 ug of bovine lactoferrin (LfB, Sigma) was used. The paper disc was inoculated with 80 μl. As a result, E. coli inhibition was not observed in the non-transformed chlorella (Non-TF) as shown in Fig. 7, and inhibition of 15 mM inhibition was observed in LfB, 10.5 mM in CT7 and 9 mM in CT9.

cfu / ml; colony forming unit

<110> PARK, BOK HEE <120> Expression of bovine lactoferrin using transformed Chlorella <130> 1 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 998 <212> DNA <213> bovine <400> 1 ccatggttcg atggtgcacc atttcccagc ccgagtggtt caaatgccgc cgatggcagt 60 ggcggatgaa gaagctgggc gctccctcta tcacctgcgt ccgcagggcc ttcgcgcttg 120 agtgcattcg ggccatcgcg gagaagaagg cggacgcggt gaccctggat ggtggcatgg 180 tgtttgaggc gggccgtgac ccctacaagc tgcgccccgt tgcagcagag atctacggga 240 cgaaggagtc cccccagacc cactactacg ccgtggccgt ggtgaagaag ggcagcaatt 300 ttcagctgga ccagctgcaa ggccggaagt cctgccatac gggccttggc cgctccgctg 360 gttggatcat ccctatgggc atcttgcgcc cgtacctaag ctggacagag tccctcgagc 420 ccctccaggg agctgtggcc aagttcttct cagcaagctg cgtaccgtgc attgacagac 480 aagcataccc taacctgtgt cagctgtgca agggggaggg ggagaaccag tgcgcctgct 540 cctcacggga gccatacttc ggttactctg gtgccttcaa gtgtctgcaa gacggggctg 600 gcgacgtggc tttcgtcaag gagactacag tgttcgagaa cttgccagag aaggctgacc 660 gcgaccagta tgagttgctt tgcctgaaca acagccgcgc cccggtggat gcgttcaagg 720 aatgccacct ggcccaggtc ccgtcgcacg ccgtcgtggc gcgaagtgtg gatggcaagg 780 aggacctgat ctggaagctc ctcagcaagg cacaggagaa gtttgggaag aacaagtcgc 840 ggagctttca gctcttcggc tctccgccgg gccagcggga cctgctgttc aaggactccg 900 cactgggatt tttgcgtatt ccgtcgaagg tggactcggc gctgtacctg ggctcccgct 960 acttgaccac cctgaagaac ctcagggaat gaggatcc 998

Claims (6)

The nucleotide sequence of SEQ ID NO: 1 is the codon optimized lactoferrin gene of Chlorella vulgaris
An expression cassette having the same characteristics as the gene map of Fig. 1 including the gene of SEQ ID NO: 1, An expression vector having the same characteristics as the gene map of Figure 3, including the expression cassette of claim 2 A recombinant lactoferrin protein characterized by expression in chlorella transformed with the expression vector of claim 3

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