JP6692101B2 - Method for producing transformed earthworm and method for producing recombinant protein - Google Patents

Description

  The present invention relates to a method for producing a transformed earthworm and a method for producing a recombinant protein.

  Recombinant proteins are produced using genetic engineering mainly in microorganisms, especially Escherichia coli, but regarding the production of recombinant proteins derived from animals, they are expressed in microorganisms for sugar chain modification etc. and obtained in an active state. Therefore, it has been conventionally attempted to produce an active form using genetically modified livestock of cultured cells or higher animals (mammals) such as goats.

  However, these methods have a problem that they are complicated and costly as compared with the case of using a microorganism, and in order to efficiently produce and recover the recombinant protein, the recombinant protein produced in the body is liquid. It is considered that the most efficient method is to secrete extracellularly and recover it, but it is extremely difficult to construct a secretory recovery system other than microorganisms, so a cheap and convenient method for producing recombinant proteins has been proposed. Was wanted.

  Under such circumstances, in recent years, a method for producing a recombinant protein using earthworms has been proposed as shown in Patent Document 1.

  In this Patent Document 1, after cutting the first half of the earthworm including the gonad, regeneration is induced to form regenerated blast cells, and a recombinant gene expression vector is injected into the regenerated blast cell site of the earthworm at the early stage of regeneration to induce traits. It has been shown that a recombinant protein can be easily obtained by preparing a transformed earthworm and recovering the recombinant protein produced by this transformed earthworm together with the crushed solution of the earthworm.

Special table 2012-532604 gazette

  However, actually, the operation of directly injecting the vector into the gonad is extremely difficult and requires skill, and cannot be easily performed by anyone.

  Moreover, since the earthworms are killed when the recombinant protein is recovered, it has been impossible to continuously and efficiently produce and recover the recombinant protein.

  The present invention has been made in view of the problems of the conventional techniques as described above, and an object of the present invention is to provide a method for producing a transformed earthworm and a method for producing a recombinant protein that can extremely easily produce a recombinant protein. To do.

  The gist of the present invention will be described with reference to the accompanying drawings.

  Earthworms belonging to the Lumbricidae family are cut or incised in the lengthwise direction, and this earthworm is anesthetized, and the cutting or regeneration cutting generated by the incision while the anesthesia is operating. Of the transformed earthworm characterized by injecting an expression vector constructed so that the target gene is expressed on the surface and transforming the earthworm by introducing this expression vector into cells or genome using electroporation It relates to the creation method.

The method for producing a transformed earthworm according to claim 1 , wherein the expression vector is injected by a microinjection method.

  In addition, earthworms belonging to the earthworm earthworm (Lumbricidae) to be used for food are cut or cut in the middle of the length direction, anesthesia treatment is given to this earthworm, and the cutting or the above in the state where the anesthesia is operating An expression vector constructed so that the gene of interest is expressed on the regenerated cut surface generated by cutting is injected by the microinjection method, and this expression vector is introduced into the cell or the genome using the electroporation method to transform the earthworm. The present invention relates to a method for producing a transformed earthworm which is characterized in that it is transformed.

Further, the present invention relates to a method for producing a recombinant protein, which comprises producing a predetermined recombinant protein by using the method for producing a transformed earthworm according to any one of claims 1 to 3 .

  INDUSTRIAL APPLICABILITY Since the present invention has been described above, a transformed earthworm that expresses a gene of interest can be produced very easily, and a recombinant protein can be easily and efficiently obtained (produced) using this transformed earthworm. It is a groundbreaking method for producing transformed earthworms and a method for producing recombinant proteins.

3 is a graph showing the results of measuring luciferase activity in Example 1.

  A preferred embodiment of the present invention will be briefly described with reference to the drawings showing the operation of the present invention.

  In the present invention, the expression vector is first injected into the earthworm. This injection method is not particularly limited, and for example, using an injection device such as a microsyringe or a microinjector, each is injected into the annulus and tail of the earthworm, or the earthworm is cut at a predetermined site, and the cut surface is cut. There is a method such as injection in the vicinity.

  Subsequently, the injected expression vector is introduced into cells or the genome to transform earthworms. In the present invention, a method in which a micropore is formed in a cell using an electroporation method and an expression vector is introduced from this hole is used.

  Specifically, for example, using an electroporator, the treatment conditions are a voltage of 5 to 100 V (preferably 10 to 40 V, more preferably 28 V), a treatment time of 5 to 300 msec (preferably 10 to 120 msec, more preferably 60 msec). The pulse is set to 1 to 20 times / sec (preferably 3 to 10 times / sec, more preferably 6 times / sec).

  After introducing an expression vector into cells using this electroporation method, a transformed earthworm expressing the target gene can be obtained by breeding for a predetermined period in an environment controlled at a predetermined temperature and humidity. A predetermined recombinant protein can be obtained by using the converted earthworm.

  In the present invention, the earthworms to be transformed are not particularly limited and it is possible to use all earthworms. For example, the earthworm earthworms and Andrei earthworms and red earthworms of the family earthworms have an appropriate size of 5 to 10 cm in length. It is particularly preferable to use these because they are excellent in protein production capacity, and because they are cultivated in large numbers in Japan, are easily available, and are extremely inexpensive.

  Further, for example, the earthworms may be anesthetized before the electroporation method. By performing the anesthesia processing, the earthworms can be treated smoothly without performing the electroporation method.

  Specific examples of the present invention will be described.

  The present example relates to a system capable of obtaining an earthworm that secretes and produces a recombinant protein by introducing an expression vector into a cell or a genome, and using the earthworm, a predetermined recombinant protein can be obtained.

  Hereinafter, this example will be described in detail.

[Creation of transformed earthworm]
In the present example, the earthworms to be transformed are the earthworms (Eisenia fetida), and specifically, the ones fasted for 24 to 48 hours are used.

  More specifically, in this example, earthworms cultivated for food with heavy metal-free feed (specifically, earthworms cultivated by the method described in Japanese Patent No. 5505750) were used.

  The earthworms to be adopted are not limited to the above, and may be, for example, Eisenia andrei and red earthworms (Lumbricus rubellus) belonging to the same earthworm family.

  In this example, the earthworms were cut at a distance of about 1 cm from the tail, and the cut tail was placed in a container lined with a sterilized water-wet cloth. Specifically, under an environment of a temperature of 15 to 30 ° C. (set to 21 ° C. in this embodiment) and a humidity of 50 to 90% (set to 70% in this embodiment), 4 to 48 hours (book) They were bred for 24 hours in the examples).

  In this example, earthworms bred in this predetermined environment were left in a 3% ethanol solution (solution temperature 4 ° C.) for about 10 minutes to anesthetize the earthworms, and the expression vector was injected while the anesthesia acted. did.

  Specifically, the luciferase expression vector (pGL4.50) was injected near the regenerated cut surface of the tail of the cut earthworm using the conventional microinjection method.

  Then, this injected luciferase expression vector was introduced into the cell (or vector) of the earthworm by electroporation.

  Specifically, using an electroporator (BTX45-2007 GEMINI X2 System manufactured by BTX), electric shock was applied to earthworms under the conditions of voltage: 28 V, treatment time: 60 msec, pulse: 6 times / second, and luciferase expression. The vector was introduced into the earthworm.

  Then, earthworms were placed in an artificial weather device set to a temperature of 15 to 30 ° C. (21 ° C. in this embodiment) and a humidity of 50 to 90% (70% in this embodiment) for 24 to 72 hours (68 in this embodiment). For a period of time) and the thus-raised earthworms were obtained as transformed earthworms.

[Luciferase activity measurement]
In this Example, luciferase activity was measured to confirm whether the transformed earthworms prepared as described above were actually transformed with the luciferase expression vector. In this example, in addition to the earthworms prepared under the above-mentioned conditions, earthworms prepared by setting the voltage condition in the electroporation method to 14V (14V condition earthworms) and empty vector in which the recombinant protein (luciferase) was not expressed. Was prepared and the above-mentioned method for producing transformed earthworms was prepared as a comparison target with earthworms (control earthworms) treated in the same manner, and the luciferase activity was also measured.

  Specifically, the luciferase activity was measured by measuring the weight of each of the transformed fragment and the fragment to be compared, and then measuring each fragment with a cell lysate (Luciferase Assay System, manufactured by Promega) and a screw cap containing beads. The mixture was added to a tube, set in a cell disruption device, disrupted 5 times under a treatment condition of 4200 rpm for 20 seconds, and then centrifuged to obtain a supernatant liquid. Luciferin ( Luciferase Assay System, manufactured by Promega) was added, and luminescence was measured using a luminometer (GloMax-Multi detection System, manufactured by Promega).

  As a result, as shown in FIG. 1, only the earthworms transformed by the method shown in this Example (voltage condition in electroporation was 28 V) showed a tendency that the luminescence intensity decreased with time. From this, it was confirmed that the luciferase expression vector was introduced and transformed to express the recombinant protein.

  As described above, by the method for producing a transformed earthworm shown in this example, even an unskilled person can produce a transformed earthworm very easily. By using this transformed earthworm, the recombinant protein can be extremely easily produced. Can be obtained.

  In addition, in the present Example, a cut was made at about 1 cm from the tail, and an expression vector (luciferase expression vector) was injected near the regenerated cut surface of the cut tail of the earthworm. You may inject the expression vector into the incision and inject the expression vector into this incision, or you may inject the expression vector into a predetermined site (for example, near the tail or annulus) of the non-cleaved earthworm without performing the cleavage treatment. ..

[How to obtain recombinant protein]
The method for obtaining the recombinant protein produced by the transformed earthworm produced by the method for producing the transformed earthworm shown in the present example will be described below.

  It is conventionally known that earthworms secrete and produce proteins or peptides. Therefore, it can be said that a predetermined recombinant protein is secreted and produced also from this transformed earthworm.

  Therefore, in this example, the recombinant protein was obtained by collecting the body cavity fluid secreted by the transformed earthworm.

  Specifically, first, the transformed earthworms are washed with water to remove dirt and the like on the body surface, and then a small amount of an organic solvent (for example, ethanol) adjusted to a concentration that does not kill the earthworms or an acid or alkali is dropped. The body cavity fluid was secreted, and the secreted body cavity fluid was recovered.

  More specifically, first, the target transformed earthworms were fasted for 26 hours, and then washed with pure water to remove body surface contaminants, and then surface water was removed.

  Subsequently, 1 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 25 mM NaCl was added to a 15 ml falcon tube, and 5 prepared transformed earthworms were added thereto, and the mixture was stirred and mixed (20 sec) to remove mucus. Then, it was taken out from the Falcon tube, washed with water, and the surface water was removed again.

  Subsequently, this transformed earthworm was placed in a 15 ml Falcon tube, 5.0 μl of 0.1 M sodium acetate buffer (pH 3.5) was added, and then the transformed earthworm was again taken out from the Falcon tube and subjected to centrifugation treatment ( 4 ° C., 6000 rpm, 15 minutes), collect the supernatant after this treatment into an Eppendorf tube, centrifuge the supernatant (4 ° C., 14000 rpm, 10 minutes), and further after this treatment The clear liquid was placed in an Eppendorf tube and centrifuged again (4 ° C., 14000 rpm, 5 minutes), and the supernatant obtained by this centrifugation was recovered as a secreted liquid to obtain a recombinant protein.

  After the addition of 5.0 μl of 0.1 M sodium acetate buffer (pH 3.5), it was confirmed that the transformed earthworms taken out from the Falcon tube were washed again with water to start their activity again without being killed. That is, by collecting the secretion liquid by the above method, it is possible to continuously collect the secretion liquid without killing the transformed earthworm.

  Regarding the method of secreting body cavity fluid, in addition to the method described above, for example, heat stimulation to earthworms using hot air or an incubator to such an extent that the earthworms are not killed (low temperature stimulation of 0 ° C to 4 ° C or 30 ° C to 40 ° C). Drugs that do not kill earthworms (for example, multi-layered), given a stimulus of irradiating ultraviolet rays by exposing to sunlight, giving a stimulus of emitting strong light using an incandescent lamp, etc. ), Or other stimuli including these stimuli may be applied to secrete body cavity fluid, and the present invention is not limited to this example.

  Further, in this example, since edible earthworms were adopted as the earthworms to be transformed, in addition to the method of recovering the recombinant protein together with the secretory fluid as described above, ingestion of the transformed earthworms themselves resulted in ingestion of the recombinant protein. It is also possible to do so.

  The operation and effect of this embodiment described above will be described below.

  In the present example, since the earthworms of the family Lamiaceae are adopted as the earthworms to be transformed, they can be obtained at low cost, easily and at low cost.

  Further, in this example, when transforming an earthworm, since the expression vector injected using the electroporation method is introduced into the cell or the genome, even an unskilled person can very easily introduce the expression vector into the cell. It can be introduced and transformed into earthworms.

  Moreover, in this embodiment, since the earthworm is anesthetized before the electroporation method is applied to the earthworm and the electroporation method is performed while the anesthesia is effective, the earthworm is not violent and the work is easy.

  Further, in this example, when the recombinant protein is secreted and recovered, the transformed earthworms are not killed, so that the secretory fluid can be continuously recovered, and an efficient recombinant protein secretory recovery system can be constructed.

  Furthermore, in this example, since the edible earthworm is adopted as this earthworm, the recombinant protein is not limited to the collection and acquisition by the secretory fluid, and the recombinant earthworm is ingested as it is without injecting and recovering the transformed earthworm. It is also possible to save the labor of secretory collection.

  Also, when the secretory fluid is recovered, it can be recovered without killing the earthworms, so that the recombinant protein can be continuously produced and recovered.

  As described above, in the present example, an earthworm that produces a photoprotein that is the main component of the firefly-derived luminescent enzyme was obtained by introducing the luciferase expression vector into the earthworm, etc., and thus the main component of the firefly-derived luminescent enzyme was obtained. It becomes possible to easily and efficiently obtain the photoprotein.

  It should be noted that the present invention is not limited to this embodiment, and the specific constitution of each constituent element can be designed as appropriate.

Claims (4)

  Earthworms belonging to the Lumbricidae family are cut or incised in the lengthwise direction, and this earthworm is anesthetized, and the cutting or regeneration cutting generated by the incision while the anesthesia is operating. Of the transformed earthworm characterized by injecting an expression vector constructed so that the target gene is expressed on the surface and transforming the earthworm by introducing this expression vector into cells or genome using electroporation How to make. The method for producing a transformed earthworm according to claim 1 , wherein the expression vector is injected by a microinjection method.   Cut or make an incision along the length of earthworms belonging to the family Lumbricidae to be used for food, and subject this earthworm to anesthesia treatment, and cut or cut the anesthesia while it is operating. An expression vector constructed so that the gene of interest is expressed on the generated regenerated cut surface is injected by the microinjection method, and this expression vector is introduced into the cell or genome using the electroporation method to transform the earthworm. A method for producing a transformed earthworm, which comprises: A method for producing a recombinant protein, which comprises producing a predetermined recombinant protein using the method for producing a transformed earthworm according to any one of claims 1 to 3 .

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