TW201723172A - Shrimp lymphocytes, method of purifying the same and method for in vitro evaluating innate immune response using the same - Google Patents

Abstract

The present invention relates to shrimp lymphocyte, a method of purifying the same and method for in vitro evaluating innate immune response using the same. A body liquid sample from shrimp is subjected to a pretreatment using an anti-coagulating agent that includes glucosamine and low-concentration of sodium ions, followed by cultivation in a cell medium that includes low-concentration of sodium ions, thereby obtaining shrimp lymphocytes with higher cell viability, cell yield and purity. The shrimp lymphocytes can be applied on in vitro evaluation of innate immune response.

Description

Shrimp lymphocytes and their purification methods are used for in vitro evaluation Method for estimating innate immune response

The invention relates to an invertebrate lymphocyte and a purification method thereof, in particular to a shrimp lymphocyte and a purification method thereof and a method for evaluating an innate immune reaction in vitro by using shrimp lymphocytes.

Shrimp are invertebrates, and the immune system of invertebrates generally uses coagulation, which is a body fluid that aggregates into clumps to form a physical barrier. It acts as the first line of defense against pathogen invasion and prevents microbes. The pathogen enters the body.

Because shrimp has only innate immunity, it does not have the adaptive immunity developed by vertebrates to pathogens, and cannot produce specific memory immune cells induced after infection with specific pathogens. Develop a vaccine for shrimp.

The most effective strategy for increasing the disease resistance of shrimp is to develop immunomodulators that promote the innate immunity of shrimp. However, shrimp cell strains capable of maintaining culture for a long period of time have not yet been established.

In view of this, it is urgent to develop a primary cell of shrimp lymphocytes to provide a screening platform for in vitro ( in vitro ) screening.

Therefore, one aspect of the present invention provides a method for purifying shrimp lymphocytes by pretreating a shrimp body fluid sample with an anti-aggregating agent containing glucosamine and a low concentration of sodium ions, and then using a low concentration. Sodium ion cell culture medium is subjected to a culture step to obtain shrimp lymphocytes having high cell viability, cell yield, and cell purity.

Another aspect of the present invention provides an anticoagulant for shrimp lymphocytes comprising glucosamine and a low concentration of sodium ions, thereby inhibiting cell aggregation rate of shrimp lymphocytes and increasing cell survival rate in vitro.

Another aspect of the present invention provides a cell culture solution for shrimp lymphocytes, comprising L-15 culture solution, fetal bovine serum and low concentration of sodium ions, thereby improving cell survival rate of shrimp lymphocytes and in vitro culture. time.

Still another aspect of the present invention provides a method for assessing an innate immune response in shrimps in vitro, which comprises co-cultivating shrimp lymphocytes obtained by the above method with a sample to be tested to evaluate whether the sample to be tested has a congenital stimulation of shrimps. The activity of the immune response.

According to the above aspect of the invention, a method for purifying shrimp lymphocytes is proposed. In one embodiment, a shrimp body fluid sample is first provided, wherein the shrimp body fluid sample is derived from white shrimp ( Litopenaeus vannamei ). Next, pretreatment is performed to uniformly mix the shrimp body fluid sample with the anti-aggregating agent to form a single cell suspension, wherein the anti-aggregating agent may comprise, for example, less than 10.0 g/L of sodium chloride and 5.5 g/L of lemon. Acid, 8.0 to 10.0 g/L sodium citrate, 19.8 to 20.0 g/L glucose, and 21.563 g/L glucosamine, and the anti-aggregating agent has a pH of pH 7.0. Then, a centrifugation step is performed to remove the supernatant from the single cell suspension and obtain a cell pellet. Thereafter, the culture step is carried out, and the cell pellet is uniformly suspended and cultured in the cell culture solution to obtain shrimp lymphocytes, wherein the cell culture solution may contain, for example, a 2-fold concentration (2X) of L-15 medium, 5 g/L. Sodium chloride, 10 g/L glucose, 15% (v/v) fetal bovine serum and have a pH of pH 7.0.

According to another aspect of the present invention, a cell culture fluid for shrimp lymphocytes is provided, wherein the anti-aggregation agent may comprise, for example, less than 10.0 g/L of sodium chloride, 5.5 g/L of citric acid, 8.0 to 10.0 g. / L of sodium citrate, 19.8 to 20.0 g / L of glucose and 21.563 g / L of glucosamine, and the anti-aggregating agent has a pH of pH 7.0.

According to an embodiment of the present invention, the anti-aggregating agent may further comprise 2.5 g/L of ethylenediaminetetraacetic acid.

According to still another aspect of the present invention, a method for assessing an innate immune response in a shrimp in vitro is presented. In one embodiment, the co-cultivation step is first performed, and the shrimp lymphocytes obtained by the above method are cultured in the above cell culture solution for 30 minutes to 72 hours, wherein the cell culture solution contains or does not contain the sample to be tested. Next, the cell viability of the shrimp lymphocytes is detected, wherein the cell viability of the shrimp lymphocytes not cultured with the sample to be tested is a reference value, and the cell viability of the shrimp lymphocytes cultured with the sample to be tested is a measured value. Then, a judging step is performed, and if the measured value is higher than the reference value, it is judged that the sample to be tested has a thorn The activity of the innate immune response of shrimp.

According to an embodiment of the present invention, before the co-cultivation step, the pre-culture step is further selectively performed to culture the shrimp lymphocytes in the cell culture solution containing no sample to be tested for at least 24 hours.

A method for purifying shrimp lymphocytes according to the present invention, which comprises pretreating a shrimp body fluid sample with an anti-aggregating agent containing glucosamine and a low concentration of sodium ions, and then performing a culture step using a cell culture solution containing a low concentration of sodium ions It can effectively improve the cell survival rate of shrimp lymphocytes and increase cell yield and cell purity. The obtained shrimp lymphocytes can be used as an in vitro screening platform to evaluate the innate immune stimulating activity of the sample to be tested.

101‧‧‧ Curve

103/105‧‧‧ collection points

201‧‧‧ shrimp body

203‧‧‧Abdominal surface extension

205‧‧‧ needle

Θ‧‧‧ angle

401/403/405/407/409/411/501/503/505‧‧‧ Curve

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

FIG. 1 is a schematic view showing a collection point of a shrimp body according to an embodiment of the present invention.

Fig. 2 is a schematic view showing the extraction of shrimp body fluid at a specific angle according to an embodiment of the present invention.

Fig. 3 is a two-dimensional scattergram showing a flow cytometer of shrimp lymphocytes according to an embodiment of the present invention.

Fig. 4 is a graph showing the cell viability of the shrimp lymphocytes treated with the anti-aggregating agents prepared in Preparation Examples 1 to 5 and Comparative Example 1 according to the present invention.

Fig. 5 is a graph showing the cell viability of the shrimp lymphocytes treated with the anti-aggregating agents prepared in Preparation Examples 1 to 5 and Comparative Example 2 according to the present invention.

Fig. 6A and Fig. 6B show optical micrographs of shrimp lymphocytes which were cultured for 120 minutes in the cell culture solutions of Comparative Example 3 (Fig. 6A) and Preparation Example 7 (Fig. 6B), respectively.

As described above, the present invention provides a method for purifying shrimp lymphocytes by pretreating a shrimp body fluid sample with an anti-aggregating agent containing glucosamine and a low concentration of sodium ions, and then using a cell containing a low concentration of sodium ions. The culture medium is subjected to a culture step to obtain shrimp lymphocytes having higher cell survival rate, higher cell yield, and higher cell purity.

In other words, the "shrimp lymphocytes" referred to herein may be, for example, primary cultured lymphocytes purified from white shrimp ( Litopenaeus vannamei ), but the species of shrimps to which the present invention is applicable are not limited thereto. In general, the shrimp lymphocytes of the present invention may comprise three groups of cells of higher cell purity, namely granular cells, semigranular cells, and hyaline cells, wherein the granular spheres (granular balls) The ratio of cells can be, for example, 36%, 9%, and 55%, respectively, but the ratio of cells of the above three groups of shrimp lymphocytes is not limited to the above, and the proportion of any group of three groups of liquid cells may be more or less. It depends on the type of shrimp used.

In addition to the shrimp species affecting the resulting shrimp lymphocytes, different collection points will also affect the number of cells obtained and cell purity. The invention can obtain more cells and shrimp cell lymphocytes with higher cell purity from a specific collection point. Please refer to FIG. 1 and FIG. 2 simultaneously, wherein FIG. 1 illustrates a first embodiment according to the present invention. Schematic diagram of the shrimp body collection point of the embodiment, and FIG. 2 is a schematic view showing the extraction of the shrimp body fluid at a specific angle according to an embodiment of the present invention.

In Fig. 1, from the collection method of the collection point 103, the chest section of the shrimp body and the first pair of abdominal joints may be first opened to expose the joint of the shrimp head breast and the shrimp body 101. Then, as shown in FIG. 2, the head of the shrimp is turned downward, and the needle (not shown) is aligned with the collecting point 103 of the joint 101, and the needle is inserted at an angle of less than 45 degrees (such as θ) in FIG. 2, wherein the angle is included. θ is defined as the angle between the abdominal surface 203 of the shrimp body 201 and the needle 205. After the tip of the needle is inserted at a depth of 0.3 cm to about 0.5 cm, the shrimp body fluid (transparent or slightly blue) is slowly withdrawn. According to some embodiments of the invention, about 0.4 mL of body fluid is available from collection point 103. According to other embodiments of the invention, only about 0.2 mL of body fluid is obtained from the collection point 105.

Another influencing factor is that the shrimp body fluid component has a clot factor and a prophenoloxidase activating system in the circulating blood of the shrimp. The degranulation will cause the agglutination activity of the shrimp body fluid, and the agglutination will cause cell aggregation. The formation of a group, which in turn causes the cells to die rapidly in the test tube. In the past , in vivo studies have found that the transglutaminase (TGase) activity of shrimp body fluids induces the coagulation mechanism of plasma clotting protein (CP). Transaminase requires the presence of cofactors such as calcium ions when acting. If the transaminase gene of the shrimp is deleted, the number of bacteria in the lymph is relatively high. In addition, some studies have shown that proteolytic enzymes (such as trypsin) used to isolate mammalian cells increase the yield of cells isolated from shrimp tissue.

The term "anti-aggregating agent" as used herein means grape Glycosamine and low concentrations of sodium ions. In one embodiment, the anti-aggregating agent may comprise, for example, less than 10.0 g/L of sodium chloride, 5.5 g/L of citric acid, 8.0 to 10.0 g/L of sodium citrate, and 19.8 to 20.0 g/L. Glucose and 21.563 g/L glucosamine, and the anti-aggregating agent has a pH of pH 7.0. In one example, the anti-aggregating agent may comprise 8.2 g/L of sodium chloride, 5.5 g/L of citric acid, 8.8 g/L of sodium citrate, 19.8 g/L of glucose, and 21.563 g/L. Glucosamine, and the anti-aggregating agent has a pH of pH 7.0. In another illustration, the anti-aggregating agent may further comprise ethylenediaminetetraacetic acid to further increase the cell viability of the shrimp lymphocytes, wherein the concentration of ethylenediaminetetraacetic acid may be, for example, 2.5 g/L.

In some embodiments, the cell viability of the shrimp lymphocytes treated with the anti-aggregating agent after 30 minutes of culture may be, for example, at least 90%. In other embodiments, the cell viability of the shrimp lymphocytes treated with the anti-aggregating agent after 60 minutes of culture may be, for example, at least 85%. In other embodiments, the cell viability of shrimp lymphocytes treated with the anti-aggregating agent after 120 minutes of culture may be, for example, at least 73%.

The term "cell culture solution" as used herein means a medium containing L-15, fetal calf serum, and a low concentration of sodium ions. In one embodiment, the cell culture fluid may comprise, for example, a 2-fold concentration (2X) of L-15 medium, less than 10.0 g/L of sodium chloride, 10 g/L of glucose, and 15% (v/v). Fetal bovine serum, and the cell culture solution has a pH of pH 7.0. In an example, the concentration of sodium chloride in the cell culture solution may be, for example, 5 g/L. In another illustration, the aforementioned cell culture fluid may further comprise an antibiotic, wherein the antibiotic may comprise, for example, streptomycin, penicillin, and Amphotericin (amphotericin). In the foregoing exemplification, the aforementioned cell culture solution may optionally comprise 100 μg/mL of streptomycin, 100 I.U./mL of penicillin, and 0.25 mg/mL of amphotericin.

In some embodiments, after the shrimp lymphocytes obtained as described above are cultured for 24 hours in the above cell culture medium, the cell viability thereof can be, for example, at least 90%.

The above method can obtain shrimp lymphocytes with high cell survival rate, cell yield and cell purity, and can be further applied to evaluate the innate immune response of shrimp in vitro. In one embodiment, the aforementioned "in vitro evaluation of shrimp innate immune response" comprises the steps of performing a co-cultivation step of culturing the shrimp lymphocytes obtained above in the cell culture medium for 30 minutes to 72 hours, wherein The cell culture fluid contains or does not contain the sample to be tested. Next, the cell viability of the shrimp lymphocytes is detected, wherein the cell viability of the shrimp lymphocytes not cultured with the sample to be tested is a reference value, and the cell viability of the shrimp lymphocytes cultured with the sample to be tested is a measured value. Then, a judging step is performed, and if the measured value is higher than the reference value, it is judged that the sample to be tested has an activity of stimulating an innate immune response of the shrimp.

In other embodiments, prior to the co-cultivation step, the pre-culture step is further performed to allow the shrimp lymphocytes to be cultured in the cell culture medium containing no sample to be tested for at least 24 hours.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Purification of shrimp lymphocytes

1. Preparation Examples 1 to 6 and Preparation Comparative Examples 1 to 2

Preparation Examples 1 to 6 and Preparation Comparative Examples 1 to 2 were prepared according to Table 1, in which anti-aggregating agents of Comparative Examples 1 to 2 were prepared, and Preparation Examples 1 to 6 were several according to the present invention. The anti-aggregating agent of the examples. The above components were dissolved in sterile water, and adjusted to pH 7.0 with a 1 N aqueous sodium hydroxide solution. The bacteria were removed by filtration through a membrane having a pore size of 0.22 μm , and placed at 4 ° C until use.

2. Preparation Example 7 and Preparation Comparative Example 3

Preparation Example 7 and Preparation Comparative Example 3 was prepared according to Table 2, wherein Comparative Example 3 was prepared as a culture solution of a conventional formulation, and Preparation Example 7 is a cell culture solution according to an embodiment of the present invention. The above components were dissolved in sterile water, and adjusted to pH 7.0 with a 1 N aqueous sodium hydroxide solution. The bacteria were removed by filtration through a membrane having a pore size of 0.22 μm , and placed at 4 ° C until use.

The formulation of Preparation Example 7 and the cell culture solution for preparing Comparative Example 3 is shown in Table 2. In the preparation, glucose can be added to 800 mL of sterilized water and completely dissolved, and then sodium chloride and 2 packets of Leibovitz's L-15 powder (equivalent to 2 times concentration (2X) of L-15) are added. After the above ingredients are completely dissolved, adjust the total volume to 840 mL with sterile water, adjust the pH to 7.0 with 1N HCl, add 10 mL (1%, v/v) antibiotic, and finally add 150 mL (15%, v/v). Fetal bovine serum (FBS), and adjusted to a total volume of 1000 mL with sterile water. The prepared cell culture medium was filtered using a membrane having a pore size of 0.22 μm to remove bacteria, and placed at 4 ° C for use.

The cell culture solution of the above Preparation Example 7 and Preparation Comparative Example 3 contained 100 μg/mL of streptomycin, 100 I.U./mL of penicillin and 0.25 mg/mL of amphotericin (CORNING). ).

3. Isolation of shrimp lymphocytes

Take 10 white shrimps ( Litopenaeus vannamei ) and obtain the body fluids of the shrimp according to different collection points.

When separating shrimp lymphocytes, first, prepare at least two pumping pumps, as well as fresh sea water, to ensure that the shrimps survive. At the same time, 0.4 mL of Preparation Examples 1 to 6 and Comparative Anti-aggregation Agents of Comparative Examples 1 to 2 were taken up in different syringes to individual syringes (total volume of the syringe was 1 mL), and placed on ice.

After moving the white shrimp to the console, let it stand for 1 hour to stabilize the shrimp, pay attention to the situation of the air and the state of the shrimp, and add fresh seawater at the right time. Next, after appropriately fixing the shrimp, the shrimp belly is exposed, and as shown in Fig. 1, the body fluid of the shrimp body is collected by two collection points as shown in Figs. 103 and 105, respectively.

Please also refer to Figure 1 and Figure 2. In Fig. 1, from the collection method of the collection point 103, the chest section of the shrimp body and the first pair of abdominal joints may be first opened to expose the joint of the shrimp head breast and the shrimp body 101. Then, as shown in Fig. 2, with the shrimp head facing down, the needle (for example, 26G x 1/2" (0.45 x 13mm) (not shown) is aligned with the collection point 103 of the joint 101, as shown in Fig. 2 The needle is less than 45 degrees (such as the angle θ), wherein the angle θ is defined as the angle between the shrimp body surface extension line 203 and the needle body 205. After the needle tip is inserted at a depth of 0.3 cm to about 0.5 cm, the shrimp body fluid is slowly extracted ( It is transparent or a little blue.) About 0.4 mL of body fluid can be obtained from the collection point 103.

As for the acquisition method by the collection point 105, the acquisition method of the above-mentioned collection point 103 is the same, except that the needle is aligned with the collection point 105 of the joint 101, and the needle is lowered by an angle of less than 45 degrees (as shown in FIG. Only about 0.2 mL of body fluid can be obtained from the collection point 105.

The shrimp body fluid obtained above is uniformly mixed with the anti-aggregating agent in the syringe in a volume ratio of 1:1 to form a mixed solution. Then, 10 μL of the mixture was added, and 90 μL of a 0.5% trypan blue solution (Biological Industries) was added to calculate the cell number and cell survival rate. The lymphocytes obtained above had an average cell count of 1 × 10 ⁷ cells/shrimp. Thereafter, the cell subsets were analyzed by a commercially available flow cytometer, and the results are shown in FIG.

Please refer to FIG. 3, which shows a (FSC/SSC) two-dimensional scattergram of a FACScan flow cytometer according to an embodiment of the present invention, wherein the horizontal axis represents side scattered light (forward). Scatter; FSC) signal strength (x1,000), and the vertical axis is the side scatter (SSC) signal strength (x1,000).

As can be seen from the results of FIG. 3, the shrimp body fluid cells obtained in Example 1 contained three high-purity lymphocytes, 36% of granular cells (G), 9% of semigranular cells (SG), and 55, respectively. % transparent ball (hyaline cells; H).

Example 2: Evaluation of anticoagulant effect of anticoagulant shrimp lymphocytes

1. To evaluate the anticoagulant effect of anti-aggregating agent glucosamine or / and trypsin on lymphocytes of shrimp

In this example, 18 white shrimps were used, and the anticoagulant formulations of Preparation Examples 1 to 5 and Preparation Comparative Example 1 were used to evaluate the anticoagulation and cell viability of shrimp lymphocytes with or without glucosamine or/and trypsin. The impact.

The shrimp body fluid cells obtained from the collection point B (shown as FIG. 1 and FIG. 103) were quickly mixed with the preparation examples 1 to 5 in the syringe and the anti-aggregation agent of the comparative example 1 in a volume ratio of 1:1. After mixing and mixing into a mixture, take 10 μL of the mixture, add 90 μL of 0.5% trypan blue solution, calculate every 10 minutes. The number of cells and cell survival rate are shown in Table 3 below and Figure 4.

Please refer to Table 3 and Figure 4, which show the number of cells (Table 3) and cell viability (Table 3) after preparation of shrimp lymphocytes according to the preparation of Examples 1 to 5 and Preparation of Comparative Example 1 according to the present invention, respectively. . Curve 403, curve 405, curve 407, curve 409, and curve 411 of FIG. 4 represent the number of lymphocytes of the shrimp treated with the anti-aggregating agents of Preparation Examples 1 to 5, respectively, and curve 401 represents the antibody of Comparative Example 1. The number of lymphocytes in the shrimp treated with the agglutinating agent.

From the results of Table 3 and Figure 4, the cell survival rate was higher after treatment with the anti-aggregating agents of Preparation Examples 1 to 5 than the conventional anti-aggregating agent of Comparative Example 1 (curve 401). Curve 403, curve 405, curve 407, curve 409, and curve 411 of FIG.

Next, after the treatment with the anti-aggregating agent of Preparation Example 2, after culturing for 0 to 60 minutes, the average number of lymphocytes of 9.38× ^{10 6} cells/shrimp can be obtained, which is better than the effects of Preparation Examples 1, 3 to 5, such as Table 3 shows. Furthermore, after treatment with the anti-aggregating agent of Preparation Example 2, the cell survival rate was maintained for a long period of time, and the cell survival rate reached 90% after 20 minutes of culture, and the cell survival rate reached 70 after 60 minutes of culture. % is better than the effects of Preparation Examples 1, 3 to 5, as shown by curve 405 of FIG.

2. To evaluate the anticoagulant effect of glucosamine and/or EDTA lymphocytes against anticoagulant

In this example, 9 white shrimps were used, and the anticoagulant formulations of Preparation Examples 2 and 6 and Preparation Comparative Example 2 were used to evaluate the presence or absence of glucosamine or/and ethylenediaminetetraacetic acid (EDTA) for shrimp lymphocytes. The effect of anticoagulation and cell viability.

The shrimp body fluid cells obtained from the collection point B (shown as Fig. 1 of Fig. 1) were quickly mixed with the preparation examples 2 and 6 in the syringe and the anti-aggregating agent prepared in Comparative Example 2 in a volume ratio of 1:1. After mixing and mixing into a mixed solution, 10 μL of the mixed solution was added, and 90 μL of a 0.5% trypan blue solution was added to calculate the cell number and cell survival rate every 10 minutes. The results are shown in Fig. 5.

Referring to Figure 5, there is shown cell viability after treatment of shrimp lymphocytes in accordance with the preparation of Examples 1 to 5 of the present invention and the preparation of Comparative Example 2. Curves 501 and 503 of Fig. 5 represent the number of lymphocytes of the shrimp treated with the anti-aggregating agents of Preparations 2 and 6, respectively, and curve 505 represents the lymphocytes of the shrimp treated with the anti-aggregating agent of Comparative Example 2. number. The anti-aggregating agent of Preparation Example 6 was further added with EDTA in the anti-aggregating agent of Preparation Example 2.

As is apparent from the results of Fig. 5, the cell survival rate was higher after the treatment with the anti-aggregating agents of Preparation Examples 2 and 6 as compared with the conventional anti-aggregating agent of Comparative Example 2, as shown by the curve 503 and the curve 501. Next, the cell viability (curve 501) after treatment with the anti-aggregating agent of Preparation Example 6 was superior to the anti-aggregating agent of Preparation Example 2 (curve 503), wherein the anti-aggregating agent of Preparation Example 6 was used. After the treatment, the cell survival rate after incubation for 30 minutes reached 90%, and the culture was carried out for 60 minutes. After that, the cell survival rate was 85%, and the cell survival rate after the culture for 120 minutes reached 73%. Therefore, the anti-aggregating agent of Preparation Example 6 was more effective, and the cells were maintained for a longer period of time and higher survival rate.

Example 3: Evaluation of the culture effect of cell culture solution on the lymphocytes of primary cultured shrimp

In this example, 20 white shrimps were used, and shrimp lymphocytes were obtained according to the method of Example 1, and immediately placed in two tubes containing 20 mL of Preparation Example 7 and Prepared Comparative Example 3 cells within 10 minutes after leaving the living body. The medium is incubated and the cells are evenly dispersed.

After counting the cells according to the above method, 10 mL of the cells were seeded in a 10 cm dish at a cell concentration of 1 × 10 ⁶ cells/mL, shaken slowly until uniform, and placed at room temperature at 25 ° C until they were attached. Thereafter, after 24 hours, 36 hours, and 72 hours of culture, the results are shown in FIGS. 6A and 6B.

Please refer to FIG. 6A and FIG. 6B, which respectively show optical micrographs of shrimp lymphocytes cultured in the cell culture medium of Comparative Example 3 (FIG. 6A) and Preparation Example 7 (FIG. 6B) for 120 minutes. From the results of Figs. 6A and 6B, the cell viability of the shrimp lymphocytes cultured in the cell culture solution of Preparation Example 7 was as high as 90% at 24 hours, as shown in Fig. 6B. In contrast, the shrimp lymphocytes cultured in the cell culture medium prepared in Comparative Example 3 were less than ideal, and all died in 24 hours, as shown in Fig. 6A.

In summary, the above-mentioned several examples demonstrate that the purification method of the shrimp lymphocytes of the present invention succeeds in using a glucosamine and a low concentration of sodium ion anti-aggregating agent to prepare the shrimp body fluid sample from a specific collection point. After the treatment, the cell culture solution containing a low concentration of sodium ions is used to carry out the culture step, thereby effectively improving the cell survival rate of the shrimp lymphocytes and increasing the cell yield and cell purity. The obtained shrimp lymphocytes can be further used as an in vitro screening platform to evaluate the innate immune stimulating activity of the sample to be tested.

For example, when the shrimp lymphocytes are used as an in vitro screening platform, the co-cultivation step may be performed first, and the shrimp lymphocytes obtained above are cultured in the above cell culture solution for 30 minutes to 72 hours, wherein the cell culture solution contains or not Contains the sample to be tested. Next, the cell viability of the shrimp lymphocytes is detected, wherein the cell viability of the shrimp lymphocytes not cultured with the sample to be tested is a reference value, and the cell viability of the shrimp lymphocytes cultured with the sample to be tested is a measured value. Then, a judging step is performed, and if the measured value is higher than the reference value, it is judged that the sample to be tested has an activity of stimulating the innate immune response of the shrimp. In other embodiments, prior to the co-cultivation step, the pre-culture step is further performed to allow the shrimp lymphocytes to be cultured in the cell culture medium containing no sample to be tested for at least 24 hours.

It should be noted that the present invention is exemplified by a specific process, a specific formulation reagent, a specific analytical method or a specific instrument, and illustrates the shrimp lymphocyte of the present invention and a purification method thereof, and a method for evaluating an innate immune reaction in vitro. However, it is to be understood by those skilled in the art that the present invention is not limited thereto, and the shrimp lymphocytes of the present invention and the purification method thereof are used for evaluating innate immune responses in vitro without departing from the spirit and scope of the present invention. The method can also be carried out using other processes, other formulated reagents, other analytical methods or other instruments.

It can be seen from the above examples that the shrimp lymphocytes of the present invention and The purification method and the method for evaluating the innate immune reaction in vitro have the advantages that the hemolysin recombinant protein (rSly) of Streptococcus suis is used as a biotype adjuvant, and the animal vaccine composition can be prepared by combining antigens of other animal diseases, Inducing antibodies in at least one of the immunized animals, while stimulating cellular and humoral immune responses to protect the immunized animal from pathogen infection.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

501/503/505‧‧‧ Curve

Claims (15)

A method for purifying shrimp lymphocytes, comprising providing a shrimp body fluid sample, wherein the shrimp body fluid sample is derived from white shrimp ( Litopenaeus vannamei ); performing a pretreatment to uniformly mix the shrimp body fluid sample with an anti-aggregating agent, To form a single cell suspension, wherein the anti-aggregating agent comprises less than 10.0 g/L of sodium chloride, 5.5 g/L of citric acid, 8.0 to 10.0 g/L of sodium citrate, 19.8 to 20.0 g/ L-glucose and 21.563 g/L glucosamine, and the anti-aggregating agent has a pH of pH 7.0; performing a centrifugation step to remove one of the supernatants of the single cell suspension and obtain a cell pellet ( Cell pellet); and performing a culture step of uniformly suspending the cell pellet and culturing it in a cell culture solution to obtain shrimp lymphocytes, wherein the cell culture solution contains a 2-fold concentration of L-15 medium, lower than 10 g/L of the sodium chloride, 10 g/L of the glucose, and 15% (v/v) of the fetal bovine serum, and the cell culture solution has the pH of pH 7.0. The method for purifying shrimp lymphocytes according to claim 1, wherein the concentration of the sodium chloride of the anti-aggregating agent is 8.2 g/L, and the concentration of the sodium citrate is 8.8 g/L, and The concentration of glucose was 19.8 g/L. The method for purifying shrimp lymphocytes according to claim 1 or 2, wherein the anti-aggregating agent further comprises 2.5 g/L of ethylenediaminetetraacetic acid. A method for purifying shrimp lymphocytes according to claim 1, wherein the concentration of the sodium chloride is 5 g/L. The method for purifying shrimp lymphocytes according to claim 1, wherein the shrimp cell lymphocyte has a cell viability of at least 90% after being cultured in the cell culture solution for 24 hours. An anti-aggregating agent for shrimp lymphocytes, comprising less than 10.0 g/L of sodium chloride, 5.5 g/L of citric acid, 8.0 to 10.0 g/L of sodium citrate, 19.8 to 20.0 g/L of glucose, and 21.563 g/L of glucosamine, and the anti-aggregating agent has a pH of pH 7.0. An anticoagulant for shrimp lymphocytes according to claim 6, wherein the concentration of the sodium chloride is 8.2 g/L, the concentration of the sodium citrate is 8.8 g/L, and the concentration of the glucose is 19.8g / L. The anticoagulant for shrimp lymphocytes according to claim 6 or 7, wherein the anti-aggregation agent further comprises 2.5 g/L of ethylenediaminetetraacetic acid. An anti-aggregating agent for shrimp lymphocytes according to claim 6, wherein one of the shrimp lymphocytes treated with the anti-aggregating agent has a cell viability of at least 90% after 30 minutes of culture. An anti-aggregating agent for shrimp lymphocytes according to claim 6, wherein one of the shrimp lymphocytes treated with the anti-aggregating agent has a cell viability of at least 85% after being cultured for 60 minutes. A cell culture solution for shrimp lymphocytes comprising a 2-fold concentration of L-15 medium, less than 10.0 g/L of sodium chloride, 10 g/L of glucose, 15% (v/v) of fetal bovine serum and having pH 7.0 pH. A cell culture solution of shrimp lymphocytes according to claim 11 of the patent application, wherein the concentration of the sodium chloride is 5 g/L. Shrimp lymph as described in claim 11 The cell culture medium of the cells, wherein one of the shrimp lymphocytes is cultured in the cell culture medium for 24 hours, and the cell survival rate is at least 90%. An in vitro method for assessing an innate immune response in a shrimp comprises: performing a co-cultivation step of culturing a shrimp lymphocyte in a cell culture medium for 30 minutes to 72 hours, wherein the cell culture solution comprises a 2-fold concentration of L- 15 culture solution, less than 10 g/L of the sodium chloride, 10 g/L of the glucose, and 15% (v/v) of the fetal bovine serum, the cell culture solution having the pH value of pH 7.0, and the The cell culture solution contains or does not include a sample to be tested; and detects a cell survival rate of the shrimp lymphocyte, wherein the cell survival rate of the shrimp lymphocyte not cultured with the sample to be tested is a reference value, and The cell survival rate of the shrimp lymphocytes cultured in the sample to be tested is a measured value; and a judging step is performed, and if the measured value is higher than the reference value, determining that the sample to be tested has an activity of stimulating the innate immune response of the shrimp . The method for assessing an innate immune response of a shrimp in vitro according to claim 14 of the patent application, before the co-cultivating step, further comprising: performing a pre-culture step of cultivating the shrimp lymphocyte without the sample to be tested The cell culture medium is for at least 24 hours.