KR100201354B1 - Process for growth promotion of flounder by oral administration of recombinant bovine growth hormone - Google Patents

Abstract

The present invention relates to a method for promoting the growth of flounder by oral administration of recombinant bovine growth hormone, and according to the method of oral administration of the mixed bovine growth hormone in the form of lyophilized powder in the feed immediately before administration Can promote growth.

Description

Flounder growth promoting method by oral administration of recombinant bovine growth hormone

1 is a graph showing the weight gain of flounder larvae following oral administration of high purity recombinant bovine growth hormone.

2 is a graph showing the increase in the length of the flounder fry after oral administration of high-purity recombinant bovine growth hormone.

3 is a graph showing the weight gain of flounder rearing fish following oral administration of high purity recombinant bovine growth hormone.

4 is a graph showing the increase in the length of the flounder fish after oral administration of high purity recombinant bovine growth hormone.

5 is a graph showing the weight gain of flounder larvae by oral administration of low purity recombinant bovine growth hormone.

6 is a graph showing the increase in the length of the flounder larvae by oral administration of low-purity recombinant bovine growth hormone.

Figure 7 is a graph showing the weight gain of flounder rearing fish following oral administration of low purity recombinant bovine growth hormone.

8 is a graph showing the increase in the length of the flatfish rearing fish following oral administration of low-purity recombinant bovine growth hormone.

The present invention relates to a method for promoting the growth of flounder by oral administration of recombinant bovine growth hormone.

Due to the limited amount of fish harvested in the natural state, aquaculture is currently increasing significantly as a solution. Economically, growth hormone is used as a method to increase the feed conversion efficiency of fish. Efforts to see are ongoing.

Efforts to promote salmon growth using purified growth hormone from mammalian pituitary glands have been attempted by Hicks et al. For the first time in 1975 (Higgs, D., Donaldson, EM, Dye, HM, and McBraide, J. R, (1975), A preliminary investigation of the effect of growth hormone on growth and muscle composition of coho salmon (Oncorhynchus kisutch), Gen. Comp.Endocrinol. 27, 240-257), growth hormones in mammals and fish isolated from the pituitary gland Many studies have been conducted on how to promote the growth of fish. However, the method of extracting and using growth hormone from the pituitary gland of mammals and fish has not been economical due to the limitation of raw material resources and the insignificant amount of extract.

On the other hand, after the first analysis of salmon growth hormone cDNA by Secain et al. (Sekine, S., Mizukami, T., Nishi, T., Kuwana, Y., Saito, A, Sato M., Itoh, S ,, and Kawauchi, H., Cloning and expression of cDNA for salmon growth hormone in Escherichia coli., Proc. Natl. Acad. Sci. USA 82, 4306-4310 (1985)), genes of several types of fish growth hormones Has been cloned, and economical mass production of growth hormone has become common by genetic engineering using microorganisms such as E. coli.

With the development of this technology, the method of promoting growth of fish using growth hormone has become more practical, and how to economically administer growth hormone to fish has emerged as an important issue.

There are three main methods of administering growth hormone to fish: injection, bathing and oral administration. Subcutaneous injection and intramuscular injection as an injection method can reliably administer growth hormone, but it is not economical because a large manpower is required. Moriyama, S. and Kawauchi, H. (1990), Growth stimulation of juvenile salmonids by immersion in recombinant salmon growth hormone, Nippon Suisan for the delivery of growth hormone by osmotic shock during immersion Gakkaishi 56, 31-34) has proved its potential, but in small cases such as larvae, it is somewhat practical, but in the case of rearing fish, the effect is not confirmed and there is a problem in economic terms. On the other hand, there are many experimental results that show that fish can be orally administered proteins or peptides unlike mammals (Pickford, GE and Atz, JW, The physiology of the pituitary gland of fishers, New York: New York Zoological Society) 1957): Asy, R ,, Protein digestion and absorption, In. Nutrition and feeling in Fish (Cowey, C, B., Macki, AM ,, Bull, JG, eds), 69-93, London, Academic Press (1985) ): McLean, E. and Donaldson, EM, Absorption of bioactive proteins by the gastrointestinal tract of fish: a review.J. Aquatic Animal Health 2, 1-11 (1990)), thus delivering growth hormone by oral administration Possibilities have been foreseen early. Donaldson, EM, Fagerulund, UH M ,, Higgs, D., and McBride, JR, In: Hormonal Enhancement of growth in fish (Hoar, WS, Randall, DJ, and Brett, JJ, eds.), Oral administration of mammalian pituitary gland attempts by Fish physiology, vol. VIII: 456-497, New York: Academic Press (1979)), while extremely minimal, promoted the growth of fish, Kawauchi et al. Oral administration of recombinant salmon growth hormone attempted by Horiyama, S., and Hirano, T., Oral administration of recombinant salmon growth hormone to rainbow trout, Oceanis 18 (1), 109-120 (1992)). It has proved effective in promoting trout growth.

In light of such circumstances, the present inventors have found that the present invention can promote fish growth by orally administering bovine growth hormone, which is effective as a growth promoting hormone, and recombinant technology and mass production technology have been established. It was completed.

That is, it is an object of the present invention to provide a method for promoting the growth of bovine growth hormone which can be economically mass-produced in an easy and economical form.

In order to achieve the above object, the present invention provides a method for promoting the growth of flounder by orally administering recombinant bovine growth hormone to flounder.

Hereinafter, the present invention will be described in detail.

In the present invention, purified recombinant bovine growth hormone prepared by culturing E. coli transformed by genetic engineering method to obtain a protein solution, and then concentrated, freeze-dried and pulverized, mixed in a feed form and orally administered to flatfish When the protein solution is lyophilized and used as a cake, the dried solid protein contains a large number of pores, so that it is not easy to dissolve, and thus it is difficult to be uniformly distributed. Therefore, the protein cake is preferably used in powder form by a process such as ball milling.

Recombinant bovine growth hormone that can be used in the present invention can be used at a high purity of 95% or more (hereinafter referred to as high purity), or a low purity of 50 to 80% (hereinafter referred to as low purity), for example, the applicant A method for producing bovine growth hormone from E. coli by a synthetic gene (Korean Patent No. 53943), a method for purifying bovine growth hormone expressed in E. coli (Korean Patent No. 86911), fermentation of a recombinant strain by adding an organic nitrogen source Method (Korean Patent Publication No. 93-21776), fed batch batch fermentation method of recombinant E. coli containing tryptophan promoter (Korea Patent Publication No. 93-21777), to prepare an active form of growth hormone from inclusion bodies of growth hormone Method (Korean Patent Publication No. 94-14780), a method for recovering protein inclusion bodies expressed in E. coli (Korean Patent Publication 94-14806), fermentation method for improving the productivity of recombinant protein using yeast extract (Korean Patent Publication No. 95-18436), culture method of recombinant E. coli to control the expression time of recombinant protein (Korean Patent Application No. 93- 30989), and high purity recombinant bovine growth hormone produced using methods used in the mass production method of bovine growth hormone (Korean Patent Application No. 94-40025) and the like, for example, filed December 13, 1995. Low purity recombinant bovine growth hormone prepared by a method for producing a recombinant bovine growth hormone for promoting fish growth can be used.

Recombinant bovine growth hormone may be well mixed and administered in a batch of flatfish feed such that 10 to 20 g per tonne weight of flatfish in high purity and 20 to 40 g per tonne weight of flatfish in low purity, once a week A total of four administrations can achieve the desired effect.

When the recombinant bovine growth hormone in the form of lyophilized powder is mixed with the feed, in the case of an extruded pellet (EP) feed, it is first dissolved in water or fish liver oil (at a concentration of about 8% or less), and evenly in the EP feed. Soak or spray the feed with a single dose and mix well. In the case of MP (Moisture Pellet) feed, the powder used for the biofeed dough of MP feed is mixed well with lyophilized bovine growth hormone, and then put into a machine for making MP feed to make pellet feed, or as in EP feed. It is prepared by first dissolving growth hormone in water or liver oil, and then mixing the feed in an MP pellet feed maker. At this time, if necessary, a binder or a binder may be added at the time of feed mixing, and as the binder, sodium polyacrylate, which is generally known, is preferable, and in addition, carboxymethylpropyl cellulose, hydroxypropylmethyl cellulose, hydroxymethyl cellulose Or mixtures thereof.

The flounder feed containing the recombinant bovine growth hormone made in the field as described above is preferably used immediately without a shelf life because of the possibility of deterioration. The dosage of growth hormone mixed in the feed is the average body weight measured before starting the administration and the criteria described above (10 to 20 g of recombinant bovine growth hormone per 1 ton of flounder weight for high purity and low purity). 20 to 40 g per 1 ton flounder weight). When mixing recombinant bovine growth hormone with flatfish feed, care should be taken to ensure that the growth hormones are mixed evenly and that the feed containing growth hormones is not consumed by some meat.

In addition, the method of the present invention can be characterized as having a very excellent effect without the use of an enteric (enteric) coating technology used to prevent the destruction of growth hormone protein in the stomach during oral administration, which can greatly reduce the manufacturing cost There is an advantage.

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following Examples.

The high purity recombinant bovine growth hormone used in the examples was prepared by the method described in Korean Patent Application No. 94-40025 (mass production method of bovine growth hormone), the low purity recombinant bovine growth hormone dated December 13, 1995 It was prepared by the method described in the method for producing recombinant bovine growth hormone for promoting fish growth of the same applicant.

Example 1

Growth-promoting Effect of Flatfish Poultry upon Oral Administration of High Purity Recombinant Bovine Growth Hormone

Each group of flatfish larvae was divided into 1 control group and 4 experimental groups for 60 days and kept in the land tank for 60 days. In the control group, only the EP feed and the EP feed the high-purity recombinant bovine growth hormone lyophilized powder in the 1 ton fish body. Each was administered to a total of four times, once a week to be mixed with the wound feed to be administered in the ratio of 1g, 5g, 10g and 20g respectively. For eight weeks, the average body weight and body length were measured in one week, and the results are shown in FIGS. 1 and 2.

1 is a graph showing the increase in the average body weight of flounder larvae by oral administration of high purity recombinant bovine growth hormone, and FIG. 2 is the increase in the average length of the flounder larvae by oral administration of high purity recombinant bovine growth hormone. The graph shown. As shown in FIG. 1 and FIG. 2, there was a growth-promoting effect in the experimental groups administered with growth hormone at the rate of more than 10 g per 1 ton weight of flounder, and the growth curve was drawn relatively steeply after the 3-4 administration of growth hormone. lost. In the 20g / tonne group, the growth rate was about 63% after 8 weeks compared to the control group and about 67% in the body length.The 10g / tonn group was 41% after 8 weeks compared to the control group. (By weight) and about 60% (by length). In the experimental group of 5 g / ton, there was no difference in body weight after 8 weeks, but the body length showed about 43% growth.

As mentioned above, the increase in the length of fish in the flounder fry was faster than the weight of the fish, which is very encouraging in light of the reality that the sales price of the flounder fry is determined by the length of the fish.

Example 2

Growth Promotion Effect of Flounder Fish from Oral Administration of High Purity Recombinant Bovine Growth Hormone

The flounders were reared in 200 fishes for 9 weeks in the control cages and the experimental groups, respectively, with 200 animals of flounder, while the control group fed only MP feed and the high-purity recombinant bovine growth hormone lyophilized powder in MP feed. A total of four doses, once a week, were mixed with the feed to be administered at a rate of 10 g and 20 g, respectively. Nine weeks were observed while measuring the average body weight and body length in 1-2 week intervals, and the results are shown in FIGS. 3 and 4.

3 is a graph showing the increase in the average fish body weight of flounder larvae by oral administration of high purity recombinant bovine growth hormone, Figure 4 is the increase in the average length of the flounder larvae by oral administration of high purity recombinant bovine growth hormone Is a graph. As shown in FIG. 3 and FIG. 4, the growth promoting effect was relatively increased after the administration of the growth hormone 4 times as in the case of Example 1. In addition, the experimental group administered the growth hormone at the rate of 10 g per 1 ton weight of flounder Showed a rapid growth of about 57% on the basis of body weight (about 41% on the body length) after 9 weeks compared to the control, and about 78% (on the basis of the body weight on the 20 g / ton experimental group after 9 weeks on the body weight). Growth was faster by about 50%). This is unusual in that the increase in weight of fish is faster than that of fish farms compared to the case of fry. In the case of flounder fish, the selling price is determined by the weight of the fish, which is very encouraging.

Example 3

Growth-promoting Effect of Flatfish by Oral Administration of Low Purity Recombinant Bovine Growth Hormone

The flounder was divided into two groups, control group 1 and experimental group 3, each for 60 days, and kept in a land tank for 60 days. The control group used only EP feed, and the experimental group added low-purity recombinant bovine growth hormone lyophilized powder to EP feed. A total of four doses, once a week, were mixed with each feed so as to be administered at a ratio of 2 g, 20 g, and 40 g of medium sugar. Eight weeks were observed while measuring the average body weight and body length on a weekly basis, and the results are shown in FIGS. 5 and 6.

5 is a graph showing the increase in the average body weight of the flounder larvae by oral administration of low-purity recombinant bovine growth hormone, Figure 6 is the increase in the average length of the flounder larvae by oral administration of low-purity recombinant bovine growth hormone Is a graph. As shown in FIG. 5 and FIG. 6, the experimental groups which received growth hormone at the rate of 20g and 40g per 1 ton weight of flounder showed a growth promoting effect and a relatively steep growth curve was drawn after the third dose of growth hormone. In the 40g / tonne experimental group, the growth rate was about 57% based on the weight of the body and about 52% of the body length after 8 weeks, compared to the control group. 47% (based on body weight) and about 48% (based on body length) were further grown. There was no difference in body weight and body length after 8 weeks in the 2 g / ton administration group compared with the control group.

As described above, when the low-purity recombinant bovine growth hormone was used for flounder fry, the effect was slightly decreased compared to the case of Example 1 using the high-purity recombinant bovine growth hormone, but not greatly reduced. In addition, in Example 1, the increase in body length was faster than the increase in body weight. In the present example, the increase in body length and body weight was similar.

Example 4

Growth Promoting Effect of Flounder Fish from Low-purity Recombinant Bovine Growth Hormone

The flounder was bred in marine cages for 9 weeks by dividing the flounder into 200 groups of control group 1 and experimental group 2, while the control group added only low-purity recombinant bovine growth hormone lyophilized powder to MP feed and 1-ton fish body. A total of four doses, once a week, were mixed with each feed so as to be administered at a ratio of 20 g and 40 g per medium. Nine weeks were observed while measuring the average body weight and body length in 1-2 weeks. The results are shown in FIGS. 7 and 8.

7 is a graph showing the weight of the average fish body weight of flounder growing fish by oral administration of low-purity recombinant bovine growth hormone, and FIG. 8 is the average length of the flounder growing fish by oral administration of low-purity recombinant bovine growth hormone It is a graph showing the increase of. As shown in FIG. 7 and FIG. 8, the growth promoting effect was relatively increased after administration of growth hormone three times as in the case of Example 3. In addition, the experimental group administered growth hormone at the rate of 20 g per 1 ton weight of the flatfish grew about 55% faster by weight (34% by length) after 9 weeks than the control group. In the case of 9 weeks, the body weight grew about 65% faster than the control group (about 41% based on the body length).

As described above, when low-purity recombinant bovine growth hormone was used for flounder breeding fish, it was observed that the increase in body weight was faster than the increase in body length as in the case of Example 2 using the high-purity recombinant bovine growth hormone. The facilitating effect tended to be slightly lower than that of high purity purified bovine growth hormone, but not significantly reduced.

As described above, the method of the present invention is not only a very small amount of recombinant bovine growth hormone mixed and administered to the feed, but also a simple oral administration method of a total of four times a week, and a difference in growth after one month of administration Regardless of the fry or rearing fish, the commercial value is very high because the effect is extremely excellent, such as about 40-80% (body weight basis) compared to the control group. In the case of flounder fry, this method can be economically beneficial for flounder cultivation, as it can shorten the time to release the seedlings by one month, which can reduce production costs and control the number of fish bottles during the fry period.

Claims (10)

A method of promoting the growth of flounder by orally administering a feed supplemented with recombinant bovine growth hormone to flounder. The method of claim 1, wherein the recombinant bovine growth hormone is in lyophilized powder form. The method of claim 1, wherein the recombinant bovine growth hormone is at least 95% pure. The method of claim 1, wherein the recombinant bovine growth hormone is 50-80% pure. The method according to claim 1, wherein the flounder is fry or rearing fish. 4. The method of claim 3, wherein the recombinant bovine growth hormone is mixed with the feed to be administered at least 10 g per tonne of weight of flounder. 5. The method of claim 4, wherein the recombinant bovine growth hormone is mixed with the feed to be administered at least 20 g per tonne of weight of flounder. The method of claim 1, wherein the recombinant bovine growth hormone is administered four times a week. The method according to claim 1, wherein the recombinant bovine growth hormone in the form of lyophilized powder is prepared in a solution state of 8% or less with water or fish liver oil, and mixed with the feed. 10. The method according to claim 1 or 9, wherein a binder for fish is used for feed mixing of the recombinant bovine growth hormone.