TW201720930A - Means and method for enhancing growth of fish - Google Patents

Abstract

The present invention provides an engineered plasmid, a recombinant protein, a recombinant Bacillus subtilis, an animal food/feed additive, a method of making recombinant Bacillus subtilis, a method of making a culture lysate, and a method of promoting growth rate of fish.

Description

Means and methods for promoting fish growth

CROSS-REFERENCE TO RELATED APPLICATIONS RELATED APPLICATIONS PCT Serial No.

The present invention relates to bioengineering means and methods for promoting the growth of fish.

Growth rate is one of the most important concerns of fish farms because of the direct relationship between growth performance and yield (and thus profits). Therefore, different attempts have been made in the industry to increase the growth rate of fish.

Fish growth hormone (FSH) has been proposed to promote fish growth. One way to promote fish growth using FSH is to soak the fish in a water bath in which FSH is dissolved. However, there are stricter restrictions on this approach. For example, in large scale, using FSH in this manner is inefficient, expensive, and impractical. Second, many fish cannot absorb FSH well in this way. Another limitation is that it is not clear whether it is safe to consume fish treated with this FSH.

The present invention seeks to solve the above problems, or at least provide the public with a useful alternative.

According to a first aspect of the present invention, there is provided a method of expressing a gene encoding fish growth hormone (tiGH) encoding a genus tilapia ( Oreochromis hornorum ) in a B. subtilis host, the method comprising the following Procedure: - Preparation of a recombinant DNA expression cassette comprising from the 5' end to the 3' end: three pairs - a veg A promoter ( veg A) and a lac operator ( lac ) present in tandem O), Bacillus subtilis shared ribosome binding site (RBS), ATG start codon, DNA sequence encoding the tiGH, and DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag) Inserting the DNA expression cassette into a shuttle vector of Bacillus subtilis or E. coli to form a recombinant construct 3vegAtiGHH6; - transforming the recombinant construct into the Bacillus subtilis host; - making the hay The Bacillus host grows in cell culture and expresses the tiGH in the cell; and - releases the tiGH by mechanically dissolving the Bacillus subtilis using a French press homogenizer

According to a second aspect of the present invention, a method for expressing a gene encoding fish growth hormone (tiGH) encoding a genus tilapia in a Bacillus subtilis host, the method comprising the steps of: - preparing a recombinant DNA expression cassette, The DNA expression cassette comprises: at least one pair of veg A promoter ( veg A) and lac operator ( lac O), Bacillus subtilis consensus ribosome binding site, ATG start codon, DNA sequence encoding the tiGH, and a DNA oligonucleotide sequence encoding a 6 histidine residue (6x His tag); - inserting the DNA expression cassette into a shuttle vector of Bacillus subtilis or E. coli to form a recombinant construct 3vegAtiGHH6; Transforming the recombinant construct into the B. subtilis host; - growing the B. subtilis host in cell culture and expressing the tiGH in the cell; and - releasing the tiGH.

Preferably, tiGH can be a single polypeptide of 22 kDa. The recombinant DNA expression cassette may include from the 5' end to the 3' end: three pairs of veg A promoter ( veg A) and lac operator ( lac O) present in tandem, Bacillus subtilis consensus ribosome binding site , ATG start codon, DNA sequence encoding the tiGH, and DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag). The shuttle vector can have a pMTLBs72 of 6.62-kb size. The transformed B. subtilis host can be grown in the presence of an IPTG inducer and/or a chloramphenicol antibiotic until the log phase is advanced or stable.

In certain embodiments, tiGH can be released mechanically from a Bacillus subtilis host, for example, by using a French pressure cell homogenizer. The method can include the step of centrifuging the cells of the B. subtilis host to obtain tiGH.

In accordance with a synergist of the present invention, there is provided a method of preparing a fish feed comprising the method of expressing a gene encoding fish growth hormone (tiGH) encoding a genus tilapia in a Bacillus subtilis host as described above.

Preferably, the cell lysate produced by cell lysis can be coated onto commercially available fish feed pellets or otherwise mixed in commercially available fish feed pellets.

In certain embodiments, the method can comprise the step of preparing a solution using an agar solution containing substantially 55.56 μl of the cell lysate per ml, wherein the cell lysate can be prepared from substantially 1.112 ml of cell culture, This cell culture produces cell lysates. The agar solution may contain substantially 0.5% sucrose and 1% agar and may be prepared substantially at a temperature of 50 °C. The mixed granules can then be dried at room temperature. The fish feed may consist essentially of 1 μg of the tiGH/1g feed.

According to a fourth aspect of the present invention, there is provided an engineered plasmid comprising a coding sequence, wherein the coding sequence comprises the following: - a) at least one pair of veg A promoter ( veg A) and lac operator ( lac O b) a consensus ribosome binding site (RBS); c) a gene encoding tilapia growth hormone (tiGH); and d) a sequence encoding a 6-histidine codon (6x His tag).

Preferably, the plasmid may contain at least three pairs of veg A promoter ( veg A) and lac operator ( lac O) operably linked together from the 5' end to the 3' end. tiGH can be the growth hormone of linalo tilapia. tiGH is operably linked to a 6-histidine codon at the 3' end of the coding sequence.

According to a fifth aspect of the invention, there is provided a recombinant protein expressed by a host in which the plasmid is integrated. Preferably, the protein can be expressed in Bacillus subtilis.

According to a sixth aspect of the present invention, there is provided a recombinant Bacillus subtilis (Bacillus subtilis) containing the plasmid construction. The recombinant Bacillus subtilis can be configured to produce genetically engineered animal food or feed additives. The recombinant Bacillus subtilis can be configured to facilitate detection and purification of tiGH expressed therefrom.

According to a seventh aspect of the present invention, there is provided an animal food or feed additive comprising the cell of the recombinant Bacillus subtilis, a lysate thereof and/or a protein expressed therefrom. The animal food or feed additive may be a fish food or a fish food additive, respectively. The animal food or feed additive may contain the cells, lysates, and/or proteins by coating the cells, lysates, and/or proteins. The animal food or feed additive may contain a lysate of the culture in which the recombinant Bacillus subtilis and an agar solution substantially melted at a ratio of 1:18 are suspended, wherein the molten agar solution comprises substantially 0.5% (w/v) )sucrose. Animal food or feed additives can be configured to feed Japanese koi for improved growth performance.

According to an eighth aspect of the present invention, a method for producing a recombinant Bacillus subtilis comprising the step of integrating the above plasmid in a wild type Bacillus subtilis is provided.

According to a ninth aspect of the invention, there is provided a method of preparing a lysate of a culture, the method comprising the step of preparing a lysate from a culture, wherein the recombinant Bacillus subtilis is grown in the culture.

According to a tenth aspect of the invention, there is provided a method of increasing the growth rate of a fish, the method comprising the step of feeding the fish using the animal feed or feed additive. The growth rate can include total weight or total length or both. The fish can be Japanese koi.

The present invention relates to the administration of fish growth hormone (FGH) to increase the growth rate of fish. For different reasons, tilapia growth hormone (tiGH) is one of the focuses of the present invention. Studies leading to the present invention have shown that recombinant forms of tiGH can not only promote the growth of tilapia's bony fish species, but also promote the growth of other fish, for example, red mullet (sockeye salmon), tilapia, goldfish, carp larvae And the larvae of carp and angelfish. More specifically, experiments during the process of the present invention demonstrate that the engineered B. subtilis (ie, GRAS) system can efficiently and efficiently express recombinant tiGH (rtiGH) having activity and functionality in the cytoplasm of a B. subtilis host, It contains a 6-histidine tag (rtiGH) at its C-terminus. Experiments were performed to confirm the identity of the resulting rtiGH, particularly by Western blot analysis and mass spectrometry. The present invention also proposes a simple method for preparing fish feed by using rtiGH produced by whole cell lysate of Bacillus subtilis containing rtiGH. Tests were conducted in randomized controlled trials to determine the efficacy of supplemental feed and normal feed for fish growth, using Japanese koi as an example of a fish model. The body weight and length of the fish were compared between the treatment group (40.38 g and 2.17 cm, respectively) and the control group (31.58 g and 1.61 cm, respectively) during the four-month study period. The conclusion was that the fish in the treated group grew significantly faster than the fish in the control group. Fish fed supplemental feeds showed healthy and well-developed, active and eager to eat at all times. See the example in Figure 10.

The following description and experiments demonstrate the invention in more detail. Materials and Methods Bacterial Strains and Media

Bacillus subtilis strain 1A751 ( eglS D102, bglT / bglS DEV, npr , apr , his ) used as a host for rtiGH expression was obtained from Bacillus Genetics Stock Centre, Ohio. E. coli strain JM101 ( supE , thi , D lac-proAB (F', tra 36, pro AB+, lacI ^q ZDM15)) used as an intermediate host for recombinant DNA work was as described above.

Bacillus subtilis transformants at 37 [deg.] C 10.5 mg l ^-1 supplemented with chloramphenicol (Fluka) in 2x LB medium (20 gl ^-1 Difco Tryptic peptone, 10 gl ^-1 Difco yeast extract, 20 gl ^-1 NaCl) Growing in. For solid medium, Bacto agar was added at a concentration of 1.5% (w/v). Recombinant plasmids were used to transform E. coli and Bacillus subtilis using the calcium chloride method and the method described by Spizizen, respectively. Species and sources of fish

(From the Mermaid Koi Center in Hong Kong) 18 18-month-old Japanese koi larvae (18-25 cm long; most: red-white) were purchased for negative experiments. On the other hand, 44 10-month-old Japanese koi larvae (mainly red and white) were donated by East River Fishing Limited (Guangdong Province, China) and were used to evaluate the biological activity of rtiGH. the study. In addition, 8 Koi fish (7 of which are 10 months old; 1 22 months old) were individually fed in a glass jar (150 cm x 43 cm x 63 cm) and fed a supplement supplemented with rtiGH. Their behavior and external characteristics were monitored within 5 months. Expression of plasmid and recombinant tiGH

The 6.62-kb Bacillus subtilis/E. coli shuttle vector pMTLBs72 was obtained from the Bacillus Genetic Stock Center. Plasmid pMTLBs72 consists of a portion of the B. subtilis pBS72 plasmid and a portion of the E. coli pBR322 sequence. It is a low copy number and stable plasmid in Bacillus subtilis with approximately 6 copies/chromosome. Using pMTLBs72 as a vector (unpublished data), the modified plasmid 3vegAcenA was constructed as before. Using 3vegAcenA, plasmid 3vegAtiGHH6 was constructed as follows. Using 14 oligo primers: P1-P14 (see sequence listing shown as SEQ ID NOs. 1-14), the first intermediate was synthesized by PCR overlap extension; the resulting product was cleaved using Spe I and Sma I, followed by The same two enzyme-restricted vectors, 3vegAcenA, were ligated to form 3vegAtiGHH6. See Figure 1.

To express rtiGH, fresh Bacillus subtilis 1A751 (3vegAtiGHH6) transformants were grown in 50 ml 2x LB medium supplemented with chloramphenicol for 8 hours at 37 °C. All cultures were then inoculated into 500 ml 2x LB medium with chloramphenicol and grown at 37 °C until the A ₆₀₀ value was equal to 1.0, followed by the addition of IPTG to a final concentration of 0.1 mM. Continue cell culture for a period of 20 hours. The culture is then centrifuged and the cell mass is lysed. Cell lysis

The retained cell pellet was dissolved in 25 ml of phosphate buffer (PB; 1.44 gl ^-1 Na ₂ HPO ₄ and 0.24 gl ^-1 KH ₂ PO ₄ ; pH 7.4) using a pressure cell homogenizer (Stansted Fluid Power Ltd., UK) Processed the resuspension 3 times. The lysate was collected for analysis and feed preparation. rtiGH analysis

Protein samples isolated on 12% (w/v) tricine-SDS-polyacrylamide gel were stained with Coomassie Brilliant Blue R250. Western blot analysis of the protein was performed using His-Probe antibody (H-3) (Santa Cruz, sc-8036). The identity of rtiGH was confirmed by liquid chromatography tandem mass spectrometry with Mascot search as previously described. Preparation of fish feed supplemented with bacterial culture lysate

In a short time of only 20 seconds, a mixture of 0.9 ml agar solution (0.5% sucrose, 1% agar; 50 ° C) and 50 μl of cell lysate containing rtiGH (concentration 100 mg l ^-1 ) showed uniform evenly Adsorbed on 5 g of fish feed (Aqua master, Koi Food, Wheat Germ, size S).

To prepare a fish feed supplemented with rtiGH, 1 g of feed was determined to contain 1 μg of rtiGH; thus 50 μl of rtiGH lysate was used to mix with 0.9 ml of agar solution. The lysate of the induced 1A751 (3vegAtiGHH6) culture was determined to contain 100 mg l ^-1 rtiGH. The protein concentration of the 1A751 (3vegAtiGHH6) lysate was determined to be 9.93 gl ^-1 , while the host lysate (negative control) had a protein concentration of 9.78 gl ^-1 . Since the same concentration of lysate protein was used to prepare various feeds, 1 g of the control feed was calibrated to contain 10 μl of host lysate. Illustrative details of a feed prepared to supplement the culture lysate are depicted in Figure 2. Finally, the coated feed was air dried at room temperature for 3 days. Evaluation of the biological activity of rtiGH

Two gray plastic sinks of the same size (168 cm x 64 cm x 16 cm) were used to feed the two groups (treatment group and control group). All variables in both slots remain the same. A recirculation system with a Qmax setting of 2,200 lh ^-1 was used to facilitate water exchange therein. The water conditions in the two tanks were kept constant as follows: temperature 23 ° C, pH 6 and dissolved oxygen 6.3 mg l ^-1 .

In a negative experiment, the fish were fed normal food pellets or supplemented with food particles of Bacillus subtilis cell lysate lacking rtiGH activity. In a randomized controlled study, fish were fed normal food pellets (control group) or food pellets (treatment group) supplemented with cell lysates containing rtiGH. Each fish sample was photographed (for identification) and weighed at predetermined time intervals and its length was measured. Results Expression of tilapia fish growth hormone in Bacillus subtilis

The DNA sequence encoding tilapia fish growth hormone (tiGH) obtained by gene assembly was cloned into the 3VegAcenA vector (unpublished data) to form a construct expressing the rtiGH 3VegAtiGHH6 (see Fig. 1). Under the induction of IPTG, Bacillus subtilis 1A751 (3VegAtiGHH6) cultures exhibited high levels of intracellular rtiGH expression. When the cell lysate of 1A751 (3VegAtiGHH6) was analyzed by SDS-PAGE, a distinct band of rtiGH (22.6 kDa) of the expected size was shown (see Figure 3). Subsequently, the band was confirmed to be tiGH by mass spectrometry (data not shown). The yield of rtiGH is estimated to be about 100 mg l ^-1 . Preparation of fish food pellets coated with Bacillus subtilis cell lysate

The conventional use of food particles added with fish growth hormone (FGH) is not new. One novel aspect of the invention relates to the specific application of Bacillus subtilis lysates to the preparation of fish feed. Although considered to be GRAS, B. subtilis cultures have an offensive odor. In order to solve the problem that the B. subtilis lysate has an odor which is not welcomed by fish, another aspect of the present invention relates to an effective method of firmly coating rtiGH suspended in a cell lysate on food particles.

The good recovery and complete band of rtiGH shown on the gel (see Figure 3) supports the conclusion that the high-pressure cell lysis method is a practical method. In order to overcome the unpleasant odor of the Bacillus subtilis lysate and the low efficiency of coating the lysate on the granules, the processability of soft agar and low level sucrose is solved. Using this procedure, the bacterial lysate is uniformly and well coated on the particles (see Figure 2). Choosing Japanese Koi as a fish model

Japanese Koi (Koi) is one of the most popular ornamental fish in China and Japan. Koi was used as a fish model in the study based on the following considerations. Koi is easy to obtain, does not picky food, grows fast, and is basically raised for viewing.

When the Koi are fed with food pellets coated with Bacillus subtilis lysate and food pellets coated with rtiGH-containing Bacillus subtilis lysate, the fish seem to enjoy both types of feed, behaving like feeding normal food pellets. the same. Effect of Bacillus subtilis lysate on the growth of Japanese koi

In order to determine whether the B. subtilis cell lysate lacking rtiGH may cause any stimulating effect on the growth of koi, a transformant Bacillus subtilis 1A751 (3VegA) containing only the 3VegA expression vector (data not shown) was first obtained. Culture lysates of B. subtilis 1A751 (3VegA) were subsequently prepared and used for coating on food particles to produce a negative control (untreated particles) for comparison. Eighteen Japanese koi were divided into two groups, one feeding untreated granules and the other feeding normal food granules. The feeding regimen of the two groups of fish (twice/day and 0.5 g/fish) was the same and the study lasted for 50 days. At the end of the study, the total weight and length of the fish were evaluated over 50 days (data not shown), there was no significant difference in growth performance, and it was concluded that the B. subtilis lysate lacking rtiGH activity against Japanese koi Growth does not have a stimulating effect. Growth stimulating activity of rtiGH

The growth stimulating effect of rtiGH was measured by the dietary supply of the following two groups of Koi. The treatment group was fed with food pellets coated with the lysate of B. subtilis 1A751 (3VegAtiGHH6) culture, while the control group was fed normal granules. The growth parameters (weight and length) of each individual Koi were measured at monthly intervals (see Figures 4 and 7). In the first two months of the study, the weight loss of most individuals in both groups (see Figure 4) may be the result of a negative reaction of the fish to various environmental effects. Later, the weight and length measurements of almost all of the fish in the two groups recorded increased. By the end of the 4 month experiment, the weight and length increments of the individuals in the treatment group showed significantly better performance than the corresponding individuals in the control group (see Figure 5, Figure 8). The overall average increase in treatment group weight was 27.9%, which was higher than the control group (see Figure 6), while the overall average increase in length of the former was 34.8%, which was higher than the latter (see Figure 9). External observation of Japanese koi feeding food pellets supplemented with rtiGH ( external view )

In order to obtain some opinions on the behavior and external characteristics of the Japanese koi (treatment group) for feeding food granules supplemented with the lysate of Bacillus subtilis containing rtiGH, eight fishes (materials and methods) were separated therefrom immediately after arrival. Individuals. These fish were then individually fed (materials and methods) in a glass jar and fed essentially only the supplemental rtiGH feed in the same manner as the treatment group.

After 5 months of feeding, all fish showed to be healthy, fleshy (and a few might even be considered short and thick), sensitive and clear in color. The fish are not afraid to stand near the observer and actively swim around the sink. More importantly, they seem to be hungry at all times, and they remain highly vigilant about food supply whenever someone passes through the sink.

The availability of fish feed with high protein content is a major cost factor to consider in fish farming. However, because feed conversion efficiency (FCE) is high in fish farming; for example, during feeding of salmon, the FCE is estimated to be in the range of 1.1 kg feed/kg salmon. Therefore, in order to improve the growth performance of fish, a good idea to flash in the mind is to increase the FCE by increasing the protein content in the fish feed. Common ingredients used to increase protein content in fish feed include fish protein hydrolysate, brine shrimp, squid viscera, soy and blue-green algae.

In scientific efforts along the same research path, recent biotechnology developments have enabled the development of transgenic fish or genetically modified (GM) fish, in which the species of the species concerned are genetically mutated, resulting in excessive endogenous growth hormone Produced to increase the growth rate of the fish. Although the FDA approved the use of GM fish for human consumption, this issue has been controversial. A good compromise with regard to the application of fish growth hormone (FGH) seems to be used as a feed supplement in fish diets.

Recombinant FGH (rFGH) from different species of fish has been successfully expressed and introduced using a variety of methods, including injection, bath, oral incubation, and feeding to fish models for evaluating their ability to promote growth under established conditions. However, in the real world, the first three methods do not seem to be practical because fish farming involves large-scale farming. Regarding the fourth method, feeding (referring to feeding the fish with food particles supplemented with rFGH) is technically feasible as long as a cost-effective way of producing the feed of interest is available. It has previously been reported that fish feed supplemented with lyophilized Synechocystis (a blue-green seaweed) supplemented with rFGH expressing olive flounder promotes the growth of halibut fish. However, despite the positive effects of feed on growth performance, this report failed to address the potential harmful effects of the microtoxin (Microcystin) present in Synechocystis on human health. This important problem combined with the relatively long time required to prepare the final additive poses a difficult challenge to the wide application of the feeding method in fish farming.

In contrast, with respect to the present invention, the rFGH expression host B. subtilis is recognized as a "GRAS" organism application. In addition, it has well-characterized genetics, relatively short doubling time (120 minutes), simple and simple processing, and most importantly, scale-up facilitating and cost-effective production supports the use of Bacillus subtilis as a host Feasibility for recombination applications. In fact, the method of preparing a fish feed (see Fig. 2; materials and methods) supplementing the whole cell lysate of B. subtilis expressing rtiGH is not only convenient but also easy to scale up. Whether the supply is small or large, the supplemental feed, like its normal counterpart, is easy to dispense and close to the fish being fed.

Prior to the experiment, a relatively long period of four months (excluding the first two months of consumption on the negative experiment) was planned for the experiment. It was subsequently determined that the observed fish may show an uncomfortable response due to new environmental and measurement procedures, affecting their appetite and leading to misinterpretation of the conclusions drawn between dietary composition and growth performance in the study. As predicted, most individuals in the treatment group (feeding supplemented rtiGH; see Figure 4A) and the control group (feeding normal feed; see Figure 4B) appear to exhibit new arrivals and/or subsequent measurements of them. It was determined that an unexpected reaction occurred. Interestingly, these side effects only affect the body weight of the two groups of fish (see Figure 4), but do not affect the body length (see Figure 7). In addition, fish weight loss was most pronounced during the first and second months of the study. Later, fish appeared to adapt to their environment and regain interest in food, thus enabling us to correlate between feed consumption and fish weight/length.

The results of the above discussion support the following conclusions. First, the two groups of koi fish consume a certain period of time to adapt to the environment and/or to introduce them newly. Second, each fish responds differently to the new environment and/or new operational processes, so it is necessary to monitor the response of individual fish. Third, due to the time required for adaptation, it was difficult to derive a reliable correlation between feed consumption and fish weight/length increase during the first two months of the study. Fourth, the length of the two groups of fish seems to be less sensitive to the response to the environment. However, in the third and fourth months of the study, changes in body length and body weight were closely related to each other, which led us to correlate between feed consumption and fish weight/length.

In the first two months of the study, regardless of the possible environmental factors that may cause fish weight loss in both the treated and control groups (see Figure 3), the difference in the overall average increase in body weight and length of the two groups of fish provides Deterministic support evidence for the performance between normal fish feed and feed supplemented with rtiGH. Although the members used in the two groups of fish showed heavier weight and longer length after four months of study (see Figures 5 and 8), the overall average increase in body weight and length of the treated group was 40.38, respectively. g (see Figure 6) and 2.17 cm (see Figure 9), significantly higher than the values of the untreated group, 31.58g (see Figure 6) and 1.61cm (see Figure 9). In fact, the inconsistencies between the two sets of data were significantly different, with an overall weight increase of 27.9% (see Figure 6) and an overall average increase of 34.8% (see Figure 9).

Therefore, our results provided in this application support the conclusion that rtiGH expressed in Bacillus subtilis is biologically active in promoting the growth of Japanese koi. Furthermore, the convenience of obtaining whole cell lysates of Bacillus subtilis provides a simple means for preparing fish feed supplemented with rtiGH. Continued efforts to improve tiGH expression, protein content and growth density of Bacillus subtilis and conditions for scale-up production have proven to be a cost-effective method for large-scale application in fish farming.

Growth rate is one of the most important points in fisheries because there is a direct relationship between growth performance and profitability. Fish growth hormone (FGH), a 22 kDa single-chain peptide produced by the pituitary gland, not only has a positive effect on growth, but also has pleiotropic functions, which is better for fish under adverse conditions (such as environmental stress and infection). Coping is key.

FGH of various species has been well characterized and their corresponding recombinants have been expressed in different microbial host systems for research. Among various FGHs, one of the adult tilapia, one of the adult tilapia that can be propagated in seawater and fresh water, is one of the focuses of the present invention. The natural FGH (tiGH) of tilapia is a polypeptide of 187 amino acids, which has a high level (more than 84%) homology with the FGH of tuna and yellowtail and is 66% identical to salmon and trout. TiGH is also expressed in recombinant form in Escherichia coli and Pichia pastoris. Due to the improved usability, sufficient research has been conducted on the efficacy of recombinant tiGH in affecting the growth rate of fish. In one embodiment, despite the relatively low level (66%) identity between tiGH and FGH of salmon and trout, it has been reported that purified recombinant tiGH from E. coli not only promotes the tilapia Species of Oreochromis mossambicus grow and also promote the growth of red snapper Oncorhynchus nerka . In another embodiment, recombinant tiGH prepared by Pichia pastoris exhibits enhanced growth, survival and quality of larvae and immunity of several larvae and larvae, such as tilapia, goldfish and squid larvae and squid And larvae of angel fish.

Interestingly, unlike the corresponding mammals, it has been shown that FGH can be administered orally to improve the growth performance of the fish. This strategy works well in teleost fish because their intestinal epithelium absorbs high molecular weight proteins. As expected, bioactive tiGH has been shown to be well absorbed by the fish gut to promote somatic growth. In addition, oral administration of FGH is associated with growth promotion of other species of fish, including coho salmon, Paralichtys olivaceus , Perca fiuviatilis , and giant catfish. More importantly, traces of oral administration of FGH could not be detected in the blood, intestine, muscle or liver of fish 90 minutes after administration. Therefore, oral administration of FGH appears to provide a safe method of improving the growth performance of fish.

One way to treat fish samples using FGH is by soaking the fish in a buffer solution in which FGH is dissolved. Although treated fish are significantly more rapidly growing than control fish soaked with conventional buffer solutions, this mode of administration is inefficient, labor intensive and not feasible on a large scale.

Our laboratories have been involved in the construction of effective microbial systems for the production of recombinant proteins for commercial use. In addition to the well-characterized Gram-negative bacterial E. coli, we believe that the Gram-positive bacterium Bacillus subtilis, which is not sufficiently studied like E. coli, has great potential for development as a universal recombinant host system. Due to the lack of an external membrane, B. subtilis is well suited for engineering for the secretion of heterologous proteins. In addition, B. subtilis is endotoxin-free due to the lack of an outer membrane, so its use is generally considered safe (GRAS).

The present invention pioneered the development of a B. subtilis secretion system for the recombinant production of beneficial proteins, including the human epidermal growth factor and the endo-glucanase of the fiber-decomposing bacteria ( Cellomonas fimi ). Subsequently, derivatives of the veg I promoter of Bacillus subtilis were constructed and proved to be functionally active in E. coli.

It is to be understood that certain features of the invention may be described in combination in a single embodiment. Conversely, various features of the invention, which are described in the context of a single embodiment, may be provided separately or in any suitable sub-combination for the sake of brevity. It should be noted that certain features of the embodiments are illustrated by way of non-limiting example. Moreover, those skilled in the art will understand the prior art which is not explained above for the sake of brevity. Some of the prior art are shown below and are incorporated herein by reference in their entirety.

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Lac O‧‧‧ lac operator
RBS‧‧‧ consensus ribosome binding site
tiGH‧‧ fish growth hormone veg
A‧‧‧ veg A promoter

The details and some embodiments of the present invention will now be explained with reference to the accompanying drawings in which: FIG. 1 is a schematic diagram showing an embodiment of a recombinant DNA construct according to the present invention; FIG. 2, including A and B, Photographs and graphs of one embodiment of a method of preparing a fish feed or feed additive; Figure 3, comprising A and B, images of a Western blot analysis of an exemplary Bacillus subtilis cell lysate obtained according to the present invention Figure 4, including A and B, is a graph showing experimental results of fish treated with food containing and without recombinant growth hormone prepared according to one embodiment of the present invention; Figure 5, including A and B, A diagram showing the growth of fish treated with and without the method or means according to one embodiment of the present invention; Figure 6 is a diagram showing two groups of fish treated with and without the method and means for growth according to the present invention, respectively. Figure of the overall average increase in weight; Figure 7, including A and B, for the effect of adding feed granules of growth hormone according to one embodiment of the present invention Figure 8 is a diagram showing the effect of treatment with or without the use of fish feed or fish additive according to one embodiment of the present invention on the length of fish; Figure 9 is a graph showing the use and non-use of the fish. A graph showing the overall average increase in length of two groups of fish treated according to the method or means for growing according to the present invention; and Figure 10 shows the use of growth hormone for tilapia and squid, respectively (with and without growth hormone) A comparison of the results of the treatment of trout), goldfish and scalar.

<110> NG, KA LUN ALAN <120> MEANS AND METHOD FOR ENHANCING GROWTH OF FISH <130> G02-006A <140> <141> <150> HK 15112136.3 <151> 2015-12-09 <160> 14 <170 > PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 acactagtaa aaagatatct aatttaaatt ttatttgaca aaaatgggct cgtgttgtac 60 aat 63 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 gaacaaacgc tggctgtctg tgatctgctg caattttatc acctcctttg tgaaattgtt 60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Descripti On of Artificial Sequence: Synthetic primer <400> 3 aacaatttca caaaggaggt gataaaattg cagcagatca cagacagcca gcgtttgttc 60 <210> 4 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400 > 4 cagagagctc tcaaagtccg agaagagtct ctgggcgagc aggtgcaggt gcgtgactct 60 gttgactgca atgga 75 <210> 5 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 5 cttctcggac tttgagagct ctctgcagac ggaggagcaa Cgtcagctca acaaaatctt 60 cctgcaggac ttctg 75 <210> 6 <211> 75 <212> DNA <213> Artificial S Qualence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 tgcgctgcgt ctcgtgtttg tcgatcgggc tgatgatgta atcagagttg cagaagtcct 60 gcaggaagat tttgt 75 <210> 7 <211> 75 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic primer <400> 7 atcgacaaac acgagacgca gcgcagctcg gtcctgaagc tgctgtcgat ctcctatgga 60 ctggttgagt cctgg 75 <210> 8 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 atctggttcc tcagagagga acctccagac agagagcgac tgggaaactc ccaggactca 60 accagtccat aggag 75 <210> 9 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 gaggttcctc tctgaggaac cagatttcac caaggctgtc tgagcttaaa acgggaatct 60 tgctgctgat caggg 75 <210> 10 < 211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 gtgctggagg gtgtcggtgt caggataatt ctctgcttca tcctgattgg ccctgatcag 60 cagcaagatt cccgt 75 <210> 11 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description Of Artificial Sequence: Synthetic primer <400> 11 tcctgacacc gacaccctcc agcacgctcc ttacggaaac tattatcaaa gtctgggagg 60 caacgaatcg ctgag 75 <210> 12 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 ccaccttgtg catgtccttc ttgaagcaag ccagcaattc ataagtttgt ctcagcgatt 60 cgttgcctcc cagac 75 <210> 13 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 13 ttcaagaagg acatgcacaa Ggtggagacc tacctgacgg tagctaaatg tcgactctct 60 ccagaagcaa actgc 75 <210> 14 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 agccccgggc taatgatgat gatgatgatg cagagtgcag tttgcttctg gagagagtcg 60 a 61

Lac O‧‧‧ lac operator

RBS‧‧‧ consensus ribosome binding site

Veg A‧‧‧ veg A promoter

Claims (12)

A method of expressing a gene encoding fish growth hormone (tiGH) encoding a genus tilapia ( Oreochromis hornorum ) in a Bacillus subtilis host, the method comprising the steps of: - preparing a recombinant DNA expression cassette, the DNA expression cassette from 5 The 'end to 3' end includes: three pairs of veg A promoter ( veg A) and lac operator ( lac O), Bacillus subtilis shared ribosome binding site (RBS), ATG start codon a DNA sequence encoding the tiGH and a DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag); - inserting the DNA expression cassette into a shuttle vector of Bacillus subtilis or Escherichia coli, thereby Forming a recombinant construct 3vegAtiGHH6; - transforming the recombinant construct into the B. subtilis host; - growing the Bacillus subtilis host in cell culture and expressing the tiGH in the cell; and - by dissolving the Bacillus subtilis Release the tiGH. A method for expressing a gene encoding fish growth hormone (tiGH) of a genus tilapia in a Bacillus subtilis host, the method comprising the steps of: - preparing a recombinant DNA expression cassette comprising: at least one pair of vegs A promoter ( veg A) and lac operator ( lac O), B. subtilis consensus ribosome binding site, ATG start codon, DNA sequence encoding the tiGH and encoding 6 histidine residues (6x a DNA oligonucleotide sequence of His tag; - inserting the DNA expression cassette into a shuttle vector of Bacillus subtilis or E. coli to form a recombinant construct 3vegAtiGHH6; - transforming the recombinant construct into the Bacillus subtilis host Medium; - causing the B. subtilis host to grow in cell culture and expressing the tiGH in the cell; and - releasing the tiGH. The method of claim 2, wherein the tiGH is a single polypeptide of 22 kDa. The method of claim 2, wherein the recombinant DNA expression cassette comprises from the 5' end to the 3' end: three pairs of veg A promoters ( veg A) and Lac operator ( lac O), B. subtilis consensus ribosome binding site, ATG start codon, DNA sequence encoding the tiGH, and DNA oligomeric nucleus encoding 6 histidine residues (6x His tag) Glycosidic acid sequence. The method of claim 2, wherein the shuttle vector is a 6.62-kb size pMTLBs72. The method of claim 2, wherein the transformed Bacillus subtilis host is grown in the presence of an IPTG inducer and/or a chloramphenicol antibiotic until the log phase is late or stable. The method of claim 2, wherein the method comprises the step of centrifuging the cells of the B. subtilis host to obtain the tiGH. The method of claim 1, wherein the release of the tiGH is achieved mechanically by using a French pressure cell homogenizer. A method of preparing a fish feed, the method comprising the method of claim 1 or 3. An engineered plasmid comprising: a coding sequence having at least one pair of veg A promoter ( veg A) and a lac operator ( lac O), a consensus ribosome binding site (RBS), encoding a tilapia growth hormone a gene of (tiGH) and a sequence encoding a 6-histidine codon (6x His tag), wherein the plasmid further comprises at least three pairs of veg A promoters operably linked together from the 5' end to the 3' end ( veg A) and lac operator ( lac O). A recombinant Bacillus subtilis containing the engineered plasmid of claim 10 of the patent application. An animal food or feed additive comprising the recombinant Bacillus subtilis cell of claim 11, or a lysate thereof or a protein expressed therefrom.